Myceliated vegetable protein and food compositions comprising same

ABSTRACT

Provided is a food composition which include a myceliated high-protein food product and methods to make such compositions, which are mixtures of myceliated high-protein food products and other edible materials. A food composition includes dairy alternative products, ready to mix beverages and beverage bases; extruded and extruded/puffed products; sheeted baked goods; meat analogs and extenders; baked goods and baking mixes; granola; and soups/soup bases. Food compositions also include texturized plant protein which can be used for making meat-structured plant protein meat analog or meat extender products. The food compositions have reduced undesirable flavors and reduced undesirable aromas due to use of myceliated high-protein food products as compared to use of similar high-protein material that is not myceliated.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application is a “continuation-in-part,” of copending U.S.patent application Ser. No. 16/025,365, filed Jul. 2, 2018, entitled“Methods for the Production and Use of Myceliated High Protein FoodCompositions,” which is a “continuation-in-part” of U.S. patentapplication Ser. No. 15/488,183, filed Apr. 14, 2017, entitled “Methodsfor the Production and Use of Myceliated High Protein FoodCompositions,” now U.S. Pat. No. 10,010,103, which claims the benefit ofU.S. Provisional Application No. 62/322,726, filed Apr. 14, 2016,entitled “Methods for the Production and Use of Myceliated High ProteinFood Compositions” now expired. This patent application also claims thebenefit of copending U.S. Provisional Patent Application No. 62/752,158,filed Oct. 29, 2018, entitled “Myceliated Vegetable Protein and FoodCompositions Comprising Same,” copending U.S. Provisional PatentApplication No. 62/857,642, filed Jun. 5, 2019, entitled “Methods forMaking Texturized Vegetable Protein and Food Compositions ComprisingSame,” and copending U.S. Provisional Patent Application No. 62/793,111,filed Jan. 16, 2019, entitled “Reaction Flavor Systems for FermentedVegetable Proteins,” all of which are incorporated by reference in theirentireties.

BACKGROUND OF THE INVENTION

There is a growing need for efficient, high quality and low-costplant-based high-protein food sources with acceptable taste, flavorand/or aroma profiles. However, it has proven difficult to achieve suchproducts, particularly with low cost vegetarian protein sources.

The present inventors have previously disclosed a method to prepare amyceliated high-protein food product, which includes culturing afilamentous fungus in an aqueous media which has a high level ofprotein, for example at least 20% protein by dry weight (w/w) and atleast 50 g/L protein. The fungi can include Pleurotus ostreatus,Pleurotus eryngii, Lepista nuda, Hericium erinaceus, Lentinula edodes,Agaricus blazeii, Laetiporus sulfureus and combinations thereof.

There is also a need for processes for creating vegetarian and veganproducts such as, alternative dairy alternative products, such as icecream, yogurt, milk, pudding, cheese; ready to mix beverages andbeverage bases for protein shakes and smoothies, dietary and nutritionalbeverages; extruded and extruded/puffed products, such as pasta noodles,crisps, and scoops, including breakfast cereals, sheeted baked goodssuch as tortillas, crackers, pizza crust; meat analogs and extenders,baked goods and baking mixes for breads, cookies, muffins, pancakes,waffles, donuts, brownies; bars, bars, such as breakfast bars, proteinbars, and the like; granola products; soups and soup bases, and thelike, which include plant proteins, as well as mixtures thereof thatinclude a higher level of plant proteins, and do not impart undesirableodors or tastes due to the presence of these plant proteins.

There is also a need to create process flavors, also known as reactionflavors, which include plant proteins, as well as mixtures thereof thatinclude a higher level of plant proteins, and do not impart undesirableodors or tastes due to the presence of these plant proteins. A reactionflavor is a flavor composition that can be used to impart, modify, orimprove the flavor of a number of different foods and beverages. Aprocess flavor is composed of a mixture of starting materials which aregenerally heated to yield a desired profile.

There is a need for meat-structured plant protein-derived meat extendersand meat analogs. This is best accomplished with textured versions ofplant proteins, which have the protein fiber/alignment properties tobest imitate the structure of meat. The art teaches textured soy proteinor pea protein concentrate or isolate, processed using an extruder inthe shape of rods or tubes. Textured soy or pea protein isolate isextruded into various shapes (chunks, flakes, nuggets, grains, andstrips) and sizes, exiting the nozzle while still hot and expanding asit does so. The thermoplastic proteins are heated to 150-200° C., whichdenatures them into a fibrous, insoluble, porous network that can soakup as much as three times its weight in liquids. As the pressurizedmolten protein mixture exits the extruder, the sudden drop in pressurecauses rapid expansion into a puffy solid that is then dried. As much as50% protein when dry, textured plant protein can be rehydrated at a 2:1ratio, which drops the percentage of protein to an approximation ofground meat at 16%.

However, in recent years, additional information has shown that thereare certain nutritional problems with consuming raw, cooked, orprocessed soybeans. The raw soybean is known to contain numerousanti-nutrients. Pea protein provides foods with a beany and/or bittertaste and a beany, pea aroma.

There is therefore a need for efficient, high quality and low costhigh-protein food sources with acceptable taste, flavor and/or aromaprofiles, and for processes for creating improved vegetarian and veganproducts such as, extruded and extruded/puffed products, meat analogsand extenders, all with improved odors and/or tastes due to the presenceof these plant proteins, but contain alternatives to soy protein.

BRIEF SUMMARY OF THE INVENTION

The invention provides a method to prepare a food composition orcompositions which comprise or include a myceliated high-protein foodproduct. In one step, the method includes providing a myceliated highprotein food product of the invention, e.g., a myceliated high-proteinfood product, wherein the myceliated high-protein food product is atleast 50% (w/w) protein on a dry weight basis, wherein the myceliatedhigh-protein is myceliated by an aqueous fungal culture, in a mediacomprising at least 50% protein in liquid culture; then, in anotherstep, providing an edible material, and mixing the myceliated highprotein food product of the invention and the edible material to formthe food composition. In embodiments, the fungal culture comprisesLentinula edodes, Agaricus spp., Pleurotus spp., Boletus spp., orLaetiporus spp.; and the myceliated high-protein food product is derivedfrom a plant source and has reduced undesirable flavors and reducedundesirable aromas compared to the high-protein material that is notmyceliated. In embodiments, the food composition is a texturized plantprotein which can be used in making, for example, meat-structured plantprotein meat analog or meat extender products.

The present invention also includes food compositions made by theinvention, and food compositions that comprise a myceliated high-proteinfood product, wherein the myceliated high-protein food product is atleast 50% (w/w) protein on a dry weight basis, wherein the myceliatedhigh-protein is myceliated by an aqueous fungal culture, in a mediacomprising at least 50% protein in liquid culture; and an ediblematerial.

Alternatively, the method for preparing food compositions which compriseor include a myceliated high-protein food product, wherein the step ofproviding a myceliated high protein food product can include thefollowing steps. In one embodiment, the myceliated high protein foodproduct is produced by culturing a fungus in an aqueous media whichincludes a high-protein material, with amounts of protein of at least50% (w/w) protein on a dry weight basis, and wherein the media comprisesat least 50 g/L protein, inoculating with a fungal culture, andculturing the medium to produce the myceliated high protein foodproducts whereby the flavor or taste of the myceliated high-protein foodproduct is modulated compared to the high-protein material; providing anedible material; and mixing the myceliated high protein food product andthe edible material to form the food composition. In embodiments, thefungal culture comprises Lentinula edodes, Agaricus spp., Pleurotusspp., Boletus spp., or Laetiporus spp.; and the myceliated high-proteinfood product is derived from a plant source and has reduced undesirableflavors and reduced undesirable aromas compared to the high-proteinmaterial that is not myceliated. In embodiments, the food composition isa texturized plant protein which can be used in making, for example,meat-structured plant protein meat analog or meat extender products. Thepresent invention also includes food compositions made by the invention.

The present invention also discloses a texturized plant protein productcomprising an extruded mixture comprising (a) a myceliated high-proteinfood product, wherein the myceliated high-protein food product is atleast 50% (w/w) protein on a dry weight basis, wherein the myceliatedhigh-protein food product is derived from pea and/or rice, wherein themyceliated high-protein product is myceliated by an aqueous fungalculture comprising Lentinula edodes, Agaricus spp., Pleurotus spp.,Boletus spp., or Laetiporus spp., in a media comprising at least 50 g/Lprotein in liquid culture, and wherein the myceliated high-protein foodproduct has reduced undesirable flavor and reduced undesirable aromacompared with a non-myceliated food product; and (b) an additionalhigh-protein material comprising at least 50% protein on a dry weightbasis, wherein the myceliated high-protein food product is present atbetween about 5% to 90% on a dry weight basis compared with theadditional high-protein material. The present invention also includestexturized plant protein products made by the invention.

The food compositions of the invention comprise, without limitation,reaction flavors, dairy alternative products, ready to mix beverages andbeverage bases; extruded and extruded/puffed products; sheeted bakedgoods; texturized plant-based protein products; baked goods and bakingmixes; granola; and soups/soup bases.

DETAILED DESCRIPTION OF THE INVENTION

The inventors have previously disclosed culturing a filamentous fungusin a high protein media to provide a protein product, and also foundthat such treatment can also alter the taste, flavor or aroma of highprotein food compositions in unexpected ways. The processes of theinvention enable the production of food compositions, proteinconcentrates, isolates and high protein foodstuffs that have been imbuedwith mycelial material, thereby altering aspects of the media used inthe production of products according to the methods of the presentinvention. The invention also presents the ability to stack proteinsources to optimize amino acid profiles of products made according tothe methods of the invention.

The invention includes methods to prepare a food composition orcompositions which comprise or include a myceliated high-protein foodproduct. In one step, the method includes providing a myceliated highprotein food product of the invention, e.g., a myceliated high-proteinfood product, wherein the myceliated high-protein food product is atleast 50% (w/w) protein on a dry weight basis, wherein the myceliatedhigh-protein is myceliated by an aqueous fungal culture, in a mediacomprising at least 50% protein in liquid culture; then, in anotherstep, providing an edible material, and mixing the myceliated highprotein food product of the invention and the edible material to formthe food composition. In embodiments, the fungal culture comprisesLentinula edodes, Agaricus spp., Pleurotus spp., Boletus spp., orLaetiporus spp.; and the myceliated high-protein food product is derivedfrom a plant source and has reduced undesirable flavors and reducedundesirable aromas compared to the high-protein material that is notmyceliated. In embodiments, the food composition is a texturized plantprotein which can be used in making, for example, meat-structured plantprotein meat analog or meat extender products.

The present invention also includes food compositions made by theinvention, and food compositions that comprise a myceliated high-proteinfood product, wherein the myceliated high-protein food product is atleast 50% (w/w) protein on a dry weight basis, wherein the myceliatedhigh-protein is myceliated by an aqueous fungal culture, in a mediacomprising at least 50% protein in liquid culture; and an ediblematerial.

Alternatively, the method for preparing food compositions which compriseor include a myceliated high-protein food product, wherein the step ofproviding a myceliated high protein food product can include thefollowing steps. In one embodiment, the myceliated high protein foodproduct is produced by culturing a fungus in an aqueous media whichincludes a high-protein material, with amounts of protein of at least50% (w/w) protein on a dry weight basis, and wherein the media comprisesat least 50 g/L protein, inoculating with a fungal culture, andculturing the medium to produce the myceliated high protein foodproducts whereby the flavor or taste of the myceliated high-protein foodproduct is modulated compared to the high-protein material; providing anedible material; and mixing the myceliated high protein food product andthe edible material to form the food composition. In embodiments, thefungal culture comprises Lentinula edodes, Agaricus spp., Pleurotusspp., Boletus spp., or Laetiporus spp.; and the myceliated high-proteinfood product is derived from a plant source and has reduced undesirableflavors and reduced undesirable aromas compared to the high-proteinmaterial that is not myceliated. In embodiments, the food composition isa texturized plant protein which can be used in making, for example,meat-structured plant protein meat analog or meat extender products. Thepresent invention also includes food compositions made by the invention.

The present invention also discloses a texturized plant protein productcomprising an extruded mixture comprising (a) a myceliated high-proteinfood product, wherein the myceliated high-protein food product is atleast 50% (w/w) protein on a dry weight basis, wherein the myceliatedhigh-protein food product is derived from pea and/or rice, wherein themyceliated high-protein product is myceliated by an aqueous fungalculture comprising Lentinula edodes, Agaricus spp., Pleurotus spp.,Boletus spp., or Laetiporus spp., in a media comprising at least 50 g/Lprotein in liquid culture, and wherein the myceliated high-protein foodproduct has reduced undesirable flavor and reduced undesirable aromacompared with a non-myceliated food product; and (b) an additionalhigh-protein material comprising at least 50% protein on a dry weightbasis, wherein the myceliated high-protein food product is present atbetween about 5% to 90% on a dry weight basis compared with theadditional high-protein material. The present invention also includestexturized plant protein products made by the invention.

In one embodiment, methods to make the textured plant-based proteinproduct includes a method step to prepare or provide a myceliatedhigh-protein food product. In order to prepare or provide a myceliatedhigh-protein food product, the method may optionally include the stepsof providing an aqueous media comprising a high-protein material. Theaqueous media may comprise, consist of, or consist essentially of atleast 50% (w/w) protein, on a dry weight basis and may also comprise atleast 50 g/L protein. The media may also comprise, consist of or consistessentially of optional additional excipients as identified hereinbelow. The aqueous media may be inoculated with a fungal culture,optionally comprising Lentinula edodes, Agaricus spp., Pleurotus spp.,Boletus spp., or Laetiporus spp. The inoculated media may then becultured to produce a myceliated high-protein food product, and themyceliated high-protein food product's taste, flavor, and/or aroma maybe modulated compared to the high-protein material in the absence of theculturing step. Alternatively, the method step to provide a myceliatedhigh-protein food product comprises providing a food product that is atleast 50% (w/w) protein on a dry weight basis, wherein the myceliatedhigh-protein food product is derived from pea and/or rice, wherein themyceliated high-protein product is myceliated by an aqueous fungalculture comprising Lentinula edodes, Agaricus spp., Pleurotus spp.,Boletus spp., or Laetiporus spp., in a media comprising at least 50 g/Lprotein in liquid culture, and wherein the myceliated high-protein foodproduct has reduced undesirable flavor and reduced undesirable aromacompared with a non-myceliated food product. The method steps to fermentand/or myceliate high protein materials to form a myceliated highprotein material are described in more detail elsewhere herein.

In an embodiment, the present invention also includes a method toprepare a textured plant-based protein product useful for products suchas meat-structured meat analogs or meat extenders. This texturedplant-based meat analog or meat extender, in one embodiment, has textureassociated with meat. The method optionally provides a “meat structuredprotein product” which can be made from the “texturized protein product”as disclosed herein. Integral to a meat structured protein product is atexturized protein product which refers to a product comprising proteinfiber networks and/or aligned protein fibers that produce meat-liketextures. It can be obtained from a dough after application of e.g.,mechanical energy (e.g., spinning, agitating, shaking, shearing,pressure, turbulence, impingement, confluence, beating, friction, wave),radiation energy (e.g., microwave, electromagnetic), thermal energy(e.g., heating, steam texturizing), enzymatic activity (e.g.,transglutaminase activity), chemical reagents (e.g., pH adjustingagents, kosmotropic salts, chaotropic salts, gypsum, surfactants,emulsifiers, fatty acids, amino acids), other methods that lead toprotein denaturation and protein fiber alignment, or combinations ofthese methods, followed by fixation of the fibrous and/or alignedstructure (e.g., by rapid temperature and/or pressure change, rapiddehydration, chemical fixation, redox), and optional post-processingafter the fibrous and/or aligned structure is generated and fixed (e.g.,hydrating, marinating, drying, coloring). Methods for determining thedegree of protein fiber network formation and/or protein fiber alignmentare known in the art and include visual determination based uponphotographs and micrographic images, as exemplified in U.S. Utilityapplication Ser. No. 14/687,803 filed Apr. 15, 2015. In someembodiments, at least about 55%, at least about 65%, at least about 75%,at least about 85%, or at least about 95% of the protein fibers aresubstantially aligned. Protein fiber networks and/or protein fiberalignments may impart cohesion and firmness whereas open spaces in theprotein fiber networks and/or protein fiber alignments may tenderize themeat structured protein products and provide pockets for capturingwater, carbohydrates, salts, lipids, flavorings, and other materialsthat are slowly released during chewing to lubricate the shearingprocess and to impart other meat-like sensory characteristics.

In one embodiment, the method to make a textured plant-based proteinproduct includes the step of providing a myceliated high-protein foodproduct and at least one additional high-protein material. In oneembodiment, the myceliated high-protein food product is at least 50%(w/w) protein on a dry weight basis, and the myceliated high-proteinfood product is derived from pea and/or rice. Additionally, themyceliated high-protein product is a myceliated high-protein productthat has been myceliated by an aqueous fungal culture, in a mediacomprising at least 50 g/L protein in liquid culture. Further, themyceliated high-protein food product has reduced undesirable flavor andreduced undesirable aroma compared with a non-myceliated food product.In this method, the additional high-protein material comprises at least50% protein on a dry weight basis.

The step of providing a textured plant-based protein product can includesteps of preparing the myceliated high-protein food product by thefollowing steps. In one embodiment, the myceliated high-protein foodproduct is produced by culturing a fungus in an aqueous media whichincludes a high-protein material, with amounts of protein of at least50% (w/w) protein, in a media comprising at least 50 g/L protein inliquid culture, resulting in a myceliated high-protein food product,whereby the flavor or taste of the myceliated high-protein food productis modulated compared to the high-protein material; providing anadditional high-protein material; and mixing the myceliated high-proteinfood product and the additional high-protein material to form a mixture;optionally preconditioning the mixture, e.g., by adding steam and/orwater to the mixture, and extruding the mixture under heat and pressureunder conditions capable of forming a textured plant-based proteinproduct useful for products such as meat-structured meat analogs or meatextenders that contain no animal products.

In the method to provide a textured plant-based protein product, themethod may also include the steps of mixing the myceliated high-proteinfood product and an additional high-protein material, wherein themyceliated high-protein food product is present at between about 5% and90% on a dry weight basis compared with the additional high-proteinmaterial. The method may also include the steps of extruding themixture. The extrusion step(s) may include the step of adding steamand/or water to the mixture and extruding the mixture under heat and/orpressure to form the textured plant-based protein product.

The method to prepare a textured plant-based protein product may alsoinclude the step of providing at least one additional high-protein foodproduct. The additional high-protein food product can comprise, consistof, or consist essentially of at least 20%, at least 25%, at least 30%,at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, or at least 95%, protein.

This additional high-protein food product, in an embodiment of themethod to make a textured plant-based protein product, has the purposeof providing gelating or other properties to supplement the formation oftexturized protein from the myceliated high-protein food product, whichin some embodiments has lower gelling properties. In an embodiment, theadditional food protein comprises a gel-forming protein. Meat isconsidered the highest quality protein source, in part because meatproteins impart specific functionalities. The overall properties of meatand meat products, including appearance, texture, and mouthfeel, aredependent on protein functionality. The principal proteinfunctionalities in processed meats are gelation and related properties(for example, meat particle binding and adhesion), emulsification, andwater-holding. Gel network formation is important to the generation oftexture within foods, providing structural matrices for holdingmoisture, flavors, sugars, and other food ingredients, and stabilizesthe dispersed phase. Gelation involves protein-protein andprotein-solvent interactions usually initiated by the unfolding of theprotein's native structure to fully extend the polypeptide chainsfollowed by intermolecular interaction to form a three-dimensionalnetwork.

In gel-forming proteins, the protein is converted to a high viscosityprogel by heat, which sets to a gel of higher viscosity upon cooling.The initial heating causes irreversible dissociation of globulinpolypeptides and subsequent heating and cooling results in a gel.

In one embodiment, in the method to make a textured plant-based proteinproduct, the present invention includes as an additional high-proteinmaterial, a gel-forming vegetarian protein. Suitable gel-formingproteins include any gel-forming protein known in the art. For thepurposes of the present invention, the additional high-protein materialis preferably a plant protein material. Suitable additional high-proteinmaterials include, without limitation, cereal proteins, including vitalwheat gluten; legume (pulse) proteins, including, without limitation,pea protein, soy protein, peanut protein, and bean proteins. Suitablegel-forming proteins may also include mycoproteins. The additionalhigh-protein material can include vegetarian sources (e.g., plantsources) as well as non-vegetarian sources, and can include a proteinconcentrate and/or isolate. Vegetarian sources include meal, proteinconcentrates and isolates prepared from a vegetarian source such aslegumes or grains, such as, including pea, rice, chickpea, soy,cyanobacteria, hemp, chia, corn gluten meal and other sources, or acombination thereof.

In an embodiment, in the method to form a textured plant-based proteinproduct, the ratio of the myceliated high-protein food product to theadditional high-protein material is between about 1% myceliatedhigh-protein food product to about 99% additional high-protein material,to about 99% myceliated high-protein food product to about 1% additionalhigh-protein material. Other ratios include about 5% myceliatedhigh-protein food product to about 95% additional high-protein material.Other ratios include about 10% to about 90%; about 15% to about 85%;about 20% to about 80%; about 25% to about 75%; about 30% to about 70%;about 35% to about 65%; about 40% to about 60%; about 45% to about 55%;about 50% to about 50%; about 55% to about 45%; about 60% to about 40%;about 65% to about 35%; about 70% to about 30%; about 75% to about 25%;about 80% to about 20%; about 85% to about 15%; about 90% to about 10%,respectively, myceliated high-protein food product to additionalhigh-protein material, w/w. In other embodiments, the amount ofmyceliated high-protein food product, relative to the additionalhigh-protein material, can vary between about 1% and 99%, between about5% and 95%, between about 10% and 90%, between about 15% and 85%,between about 20% and 80%, between about 25% and 75%, between about 30%and 70%, between about 35% and 65%, between about 40% and 60%, betweenabout 45% and 55%, or about 48% to about 52%, or, about 40% and 85%,between about 45% and 80%, between about 50% and 75%.

The method to prepare a textured plant-based protein product may alsoinclude the step of providing an optional carbohydrate component. Thecarbohydrate ingredients are typically classified as a starch, a flour,or an edible fiber and the carbohydrate component may comprise one ormore types of starch, flour, edible fiber, and combinations thereof.

Starch is the primary carbohydrate source used to help the formation ofthe product texture in textured plant-based protein products. Typicalstarches used include rice starch, wheat starch, oat starch, cornstarch, potato starch, cassava starch, and tapioca starch, althoughstarch from any source is contemplated. Overall, the swelling ability ofstarch, solubility, amount of amylose leaching out duringgelatinization, and the ability to produce a viscous paste, have aneffect on the textured plant-based protein product. Chemical alterationsoccur due to structural changes of the macromolecules in the feed blend,such as starch gelatinization and protein denaturation, as well asincorporation of water into the molecular matrix, all of which convertthe raw feed particles into a viscoelastic dough under a pressurizedenvironment. Physical changes, on the other hand, are related to productexpansion due to a drastic pressure drop and water evaporation duringdie exit.

Alternatively, the textured plant-based protein product can containstarch or flour from at least one cereal grain. Examples of flourinclude wheat flour, rice flour, barley flour white corn flour, oatflour, sorghum flour, rye flour, amaranth flour, quinoa flour, andcombinations thereof.

In one embodiment, the textured plant-based protein product includes anedible fiber. Examples of suitable edible fiber include but are notlimited to bamboo fiber, barley bran, carrot fiber, citrus fiber, cornbran, soluble dietary fiber, insoluble dietary fiber, oat bran, peafiber, soy fiber, soy polysaccharide, wheat bran, wood pulp cellulose,modified cellulose, seed husks, oat hulls, citrus fiber, carrot fiber,corn bran, soy polysaccharide, barley bran, and rice bran. The fiber maybe present in the dry pre-mix from about 0.1% to about 10% by weight. Inone embodiment, the fiber is about 2% to about 8% by weight of the dryingredients. In another embodiment the fiber is about 5% by weight ofthe dry ingredients. Particularly desirable types of fiber are thosethat effectively bind water when the mixture of non-animal protein andfiber is extruded.

In some embodiments, a textured plant-based protein product compriseswhere at least some of the carbohydrate is derived from pea. In someembodiments, the mixture comprises between about 0.1% and about 25%,between about 3% and about 20%, between about 5% and about 15%, betweenabout 5% and about 10%, between about 4% and about 7%, or between about3% and about 35% by weight of carbohydrate, such as pea fiber.

Without being bound by theory, in the present invention comprising atextured plant-based protein product, it was found that the addition offiber, including the addition of pea fiber, to the mixture, providedunique properties to the textured plant-based protein product, asdetailed in the Examples. In embodiments, the textured plant-basedprotein product includes a textured plant-based protein product formedfrom a mixture that comprises about 45% pea protein, 5% pea fiber, andabout 50% myceliated high-protein material, w/w, or a mixture thatcomprises about 70% myceliated high-protein material, about 25% peaprotein, about 5% pea fiber, w/w.

In embodiments, the moisture of the textured plant-based protein productis between about 20% and 40% by weight, and may be dried toapproximately between 3% and 5% moisture to allow for rehydration byfinished food manufacturers. In an embodiment, the dried texturedplant-based protein product rehydrates to between 0.5 and 3 times itsweight in water within a period of time after water addition, forexample, 10 minutes. In an embodiment, the textured plant protein has areduced undesirable flavor, such as, for example, a pea flavor or abitterness flavor, and a reduced undesirable aroma, such as, forexample, a beany aroma or a rice aroma.

Other ingredients in the mixture to form the textured plant-basedprotein product may include pH modifiers. Modifying pH above 8.0 alsomay result in the production of harmful lysinoalanines; and lowering thepH has the opposite effect and will decrease protein solubility, makingthe protein more difficult to process. In an embodiment, pH isconsidered between pH 6.5 and 7.5. Other ingredients may includeprocessing aids such as calcium salts, such as calcium chloride, whichcan increase the textural integrity of extruded vegetable proteins andaid in smoothing its surface. Dosing levels for CaCl₂) range between 0.5and 2.0%. Sulfur compounds are useful for their ability to aid in thecleavage of disulfide bonding, which assists the unraveling of longtwisted protein molecules. This reaction with the protein moleculescauses increased expansion, smooth product surface and adds stability tothe extrusion process, however, off flavors and aromas can arise fromtheir use. Appropriate sulfur compounds to use in the present inventionincludes sodium metabisulfite, sodium bisulfite, as well as cystine, canbe used with effects similar to those from using sulfur. The normaldosing levels for sulfur or sulfur derivatives—are in the 0.1 to 0.2%range. Cystine is used at approximately 0.5 to 1% level.

The present invention also includes products such as meat-structuredplant-based meat analogs or meat extenders, for example, a plant-basedmeat analog patty, a plant-based meat analog sausage, a plant-based meatanalog frankfurter, and a plant-based meat analog meat extended patty,sausage, or frankfurter comprising the textured plant-based proteinproduct of the present invention.

In one embodiment, transglutaminase can be used as a processing aid inthe present invention to improve textural attributes, water-holdingcapacity and appearance of the textured plant-based protein product. Inparticular, transglutaminase may improve the firmness, viscoelasticitywater binding and gelling capacity of the compositions or food products.Transglutaminase is an enzyme that catalyzes the formation ofcross-links both within a protein molecule and between molecules ofdifferent proteins. Microbial transglutaminase (MTG or MTGase) catalyzescovalent cross-link formation between primary amines (such as c aminogroup of lysine) and the amide group of glutamine residues to form highmolecular weight polymers, resulting in improvement of mechanicalproperties.

MTGase is used as a texturizing agent in several meat products, such assausages, burgers and restructured meats. In addition to the positiveimpact on the texture of the final product, transglutaminase inclusionfacilitates strong cohesion of meat blocks or multi-meat compositionswithout the need for thermal processing or addition of salt orphosphates. Use of transglutaminase in meat processing can significantlyimprove the texture of the final product, which results in, for example,an increase in its hardness. Moreover, it strengthens the texture ofhomogenized sausages made of pork, beef or poultry meat. The applicationof transglutaminase mediated textured plant-based protein product hasthe potential to offer new technological opportunities for producingrestructured meats, fine and coarse-minced sausages, fresh and frozenhamburgers, hot dogs and similar products. Transglutaminase may be usedin the textured plant-based protein products of the present invention toimprove gelation through the formation of covalent intramolecular andintermolecular bonds. For example, transglutaminase may increasehardness, springiness, chewiness, and cutting-force of the texturedplant-based meat analog of the present invention. Transglutaminase maybe used at amounts known in the art, generally up to about 0.75%.

Microbial transglutaminase (M-TG) are active over a broad range of pH4.5 to 8.0 and temperatures 0° C. to 45° C. and require calcium. Themyceliated high-protein product and/or additional high-protein material,either as is or in combination with other protein matrices (such as theadditional high-protein material) can be mixed with transglutaminase inthe range of 0.1% to 0.7% to promote crosslinking and form a solidmatrix/restructured form that can be further processed at elevatedtemperatures (>75° C. for 5 mins) to inactivate the enzyme.

Therefore, the methods of the invention further include a method to makea restructured textured plant-based protein product comprising the stepsof providing a proteinaceous material, e.g., a myceliated high-proteinfood product, and mixing with transglutaminase to form the restructuredtextured plant-based protein product. In embodiments, the proteinaceousmaterial includes an additional high-protein food product and otheringredients, excipients or additives as disclosed herein. The inventionfurther includes a restructured textured plant-based protein productmeat analog product or extender.

Seasonings can be added before or after the extruding and/or cookingand/or puffing steps. Seasonings include, but are not limited to,minerals such as salt, grain-based seasonings (such as, but not limitedto, whole, cracked or ground wheat, corn, oats, rye, flax, barley, speltand rice), plant-derived seasonings (such as, but not limited to, onion,garlic, pepper, capsicum pepper, herbs, spices, nuts, olives, fruits,vegetables, etc.), and other flavorings (such as, but not limited to,vanilla, sugar, cheese, yeast extract, whey), and combinations thereof.Vitamins can also be included such as, but not limited to, niacin, iron,zinc, thiamine mononitrate (vitamin B1), riboflavin (vitamin B2), folicacid, tocopherol(s) (vitamin E), vitamin C, vitamin B6, vitamin B12,vitamin A, vitamin D, pantothenic acid and copper. Edible oil and fatcan also be included. Oils such as, but not limited to, soy, corn,canola, sesame, safflower, olive, sunflower, rapeseed, cottonseed,peanut, copra, palm kernel, palm, linseed, lupin, and combinationsthereof can be used. Other fats such as butter or lecithin and theirmixtures can also be used. Other ingredients can be included such asemulsifiers (such as, but not limited to, lecithin, soy lecithin),leavening (such as, but not limited to, baking soda, calcium phosphate,yeast), natural and artificial sweeteners, preservatives (such as, butnot limited to, BHT, BHA, and tocopherol), fiber (such as, but notlimited to, insoluble fiber, soluble fiber (e.g., Fibersol®)), and anycombinations of such ingredients.

The term “dry ingredients” includes all the ingredients in the mixtureto form the textured plant-based protein product except for added waterand ingredients added with the added water (i.e., the “wetingredients”). Thus, the dry ingredients can include the myceliatedhigh-protein food product component, the additional high-proteinmaterial component, such as wheat gluten; the carbohydrate (e.g., fiber)component, and other processing aids as discussed herein.

In addition to the foregoing, the ingredients utilized to make thetextured plant-based protein product may include an edible lipidcomponent that comprises one or more edible lipids. In accordance withthe present disclosure, nearly any edible lipid material may beemployed, including natural and synthetic oils, for example, rapeseed,canola, soybean, coconut, cottonseed, peanut, palm and corn oils and ineither non-hydrogenated or hydrogenated form. In one embodiment, theedible lipid material is an edible vegetable oil, such as canola oil,coconut oil, cottonseed oil, peanut oil, and olive oil. In oneembodiment, the total edible lipid content is no more than about 5% ofthe weight of the dry ingredients utilized the make the meat analogproduct. As such, in one embodiment, the total edible lipid content isan amount of about 0.1% to about 1% by weight of the dry ingredients. Inanother embodiment, the total edible lipid content is an amount of about0.2% to about 0.5% by weight of the dry ingredients.

In addition to the foregoing, the textured plant-based protein productcan optionally include water at a relatively high amount. In oneembodiment, the total moisture level of the mixture extruded to make thetextured plant-based protein product is controlled such that thetextured plant-based protein product has a moisture content that is atleast about 50% by weight. To achieve such a high moisture content,water is typically added to the ingredients. Although, a relatively highmoisture content is desirable, it may not be desirable for the texturedplant-based protein product to have a moisture content much greater thanabout 65%. As such, in one embodiment the amount of water added to theingredients and the extrusion process parameters are controlled suchthat the textured plant-based protein product (following extrusion) hasa moisture content that is from about 40% to about 65% by weight. Thetextured plant-based protein product may be optionally dried to form thefinal commercial product, which is then rehydrated by the finished foodproduct manufacturer.

In an exemplary embodiment, a texturized plant protein product caninclude between about 1-99%, preferably 85% to 95%, of a combination ofa myceliated high-protein food product and an additional high-proteinmaterial; in a vegan sausage type product; using a twin screw extruder,the mixture can be further blended with starch or fiber of 5-15% (allpercentages w/w dry ingredients).

Such textured plant-based protein products can be used to create anumber of new food compositions, including, without limitation meatanalogs and extenders, which contain a myceliated high-protein foodproduct. The methods to prepare a food composition can include theadditional, optional steps of cooking, extruding, and puffing the foodcomposition according to methods known in the art to form the foodcompositions comprising the textured plant-based protein products of theinvention.

Extrusion is a technology to produce texturized proteins, a uniqueproduct which can be produced from a wide range of raw ingredientspecifications, while controlling the functional properties such asdensity, rate and time of rehydration, shape, product appearance andmouthfeel.

The general procedure is as follows. The flour mix is prepared andtypically the dry ingredients are blended together in the premixturestage. In the optional preconditioning step (in a section of an extruderdevice known as preconditioner) the steam and water are usually added atthis stage to wet/moisten and warm the flour mix. In the extruder, themajority of the work happens. Generally, the starch and protein areplasticized using heat, pressure and/or mechanical shear, then realignedand expanded as the mixture exits the extruder. The material coming fromthe extruder moisture ranges from 25% to 30%. Optionally, this extrudedmaterial can be dried to about 3%-5% moisture or less in the dryerportion. Cooling then optionally occurs to lower the temperature of thedried product to ambient conditions followed by an optional packagingstep.

Texturization of plant proteins have been a significant development inthe food processing industry. Substitution of textured plant protein(TPP) for traditional meat has several advantages in terms of cost,energy efficiency, increased protein food supply, and sustainable whencompared to conventional animal production and slaughter.

Generally, two types of textured meat-like products are derived fromtexturization processes. First is a meat extender that has been commonlyproduced by processing plant proteins through high pressure/extrusionstep. Resulting texturized product shows distinct fiber formation andhighly expanded. On rehydration the TPP can be used to extend groundmeat or meat products. Second type are meat analogs which are used inplace of meat. The TPP based meat analogs are dense, have alayered-fiber conformations and maintains meat-like character afterextensive cooking or retorting. High moisture extrusion processes havebeen used to produce meat analogs.

A typical extruder can be made up of the following parts. The Flour MixFeeder—the bin/feeder provides a means of uniformly metering the rawmaterials (granular or floury in nature) into a preconditioner or mixingcylinder and subsequently into the extruder itself. Also controls theproduct rate or throughput of the entire system. Preconditioner—heresteam and water are metered and injected into the raw material tocontrol raw material temperature and moisture. Preconditioning promotesmoisture and heat penetration of the individual particles, resulting inuniform moisture application and elevated raw material temperature. Inthe absence of a preconditioner, a longer extruder barrel with adequatemixing and hydration zones may serve the purpose of a preconditioner.Typical moisture content of the raw material exiting the preconditionerrange from 10% to 25% and temperatures in the range of 110° F. to 120°F.

The extruder portion itself is described as follows. Extruders arepopularly classified as either being a single or twin-screw design. Inboth designs, the impact on final product texture is affected by screwand barrel profile, screw speed, processing conditions such astemperature, moisture, pressure, raw material characteristics and dieselection. The initial section of the extruder barrel is designed to actas a feeding or metering zone and simply conveys the raw orpreconditioned vegetable protein away from the inlet portion of thebarrel and into the extruder. Product then enters a processing zonewhere the amorphous, free-flowing vegetable protein is worked into acolloidal dough. The compression ratio of the screw profile is increasedin this stage to assist in blending water or steam with the rawmaterial. The temperature of this moist, proteinaceous dough is rapidlyelevated in the last 2-5 seconds of dwell time within the extruderbarrel. Screw profile can be altered by the pitch, flight height andangle, and steamlock diameter, which affects the conveying of thisplasticized food material down the screw channel. The net flow patternsof the product within the screw are quite complicated and difficult tounderstand and describe.

Hydration and heating cause unravelling of the long, twisted proteinmolecules in vegetable proteins. In the extrusion process, thesemolecules align themselves along the streamline flows of the screws anddies. The increase in shear temperature and retention time causescross-linking to occur, yielding a textured product that is layered, andthe resulting denaturation or cross-linking can be considered anirreversible endothermic chemical reaction. The extent of cross-linkingis a function of the time, temperature and moisture history and can berelated to changes in apparent viscosity of the extrudate. The properexposure to shearing action, as the protein molecules align themselvesfor crosslinking during the extrusion process.

A food composition of the invention can also include a texturizedprotein, such as a texturized plant protein. Texturized plant proteincomprising the myceliated high-protein product of the present inventioninclude meat analog products and methods for making meat analog productscomprising the myceliated high-protein product as disclosed within. Themeat analog products can be produced with high moisture content andprovide a product that simulates the fibrous structure of animal meatand has a desirable meat-like moisture, texture, mouthfeel, flavor andcolor. Meat analog products having qualities (for example, texture,moisture, mouthfeel, flavor, and color) similar to that of whole muscleanimal meat may be produced using non-animal proteins formed usingextrusion under conditions of relatively high moisture. In oneembodiment, meat analog products may include myceliated high-proteinproducts of the invention, an additional high-protein material, one ormore of flour, starch, an edible lipid material and optionally a flavormodifier.

Due to its versatility, high productivity, energy efficiency and lowcost, extrusion processing is widely used in the modern food industry.Extrusion processing is a multi-step and multifunctional operation,which leads to mixing, hydration, shear, homogenization, compression,deaeration. pasteurization or sterilization, stream alignment, shaping,expansion and/or fiber formation. Ultimately, the non-animal protein,typically introduced to the extruder in the form of a dry blend, isprocessed to form a fibrous material.

It is known in the art for texturized plant protein materials, toutilize twin screw extruders under high moisture (40-80%) conditions fortexturizing non-animal proteins into fibrous meat alternatives. In thehigh moisture twin screw process, also known as “wet extrusion”, the rawmaterials, predominantly non-animal proteins such the myceliatedhigh-protein products of the invention, are mixed and fed into atwin-screw extruder, here a proper amount of water is dosed in and allingredients are further blended and then melted by the thermo-mechanicalaction of the screws. The realignment of large protein molecules, thelaminar flow, and the strong tendency of stratification within theextruder's long slit cooling die contribute to the formation of afibrous structure. The resulting wet-extruded products tend to exhibitimproved whole muscle meat-like visual appearance and improvedpalatability. Therefore, this extrusion technology shows promise fortexturizing non-animal proteins to meet increasing consumer demands forhealthy and tasty foods.

Texturization of protein is the development of a texture or a structurevia a process involving heat, and/or shear and the addition of water.The texture or structure will be formed by protein fibers that willprovide a meat-like appearance and perception when consumed. Themechanism of texturization of proteins starts with the hydration andunfolding of a given protein by breaking intramolecular binding forcesby heat and/or shear. The unfolded proteins molecules are aligned andbound by shear, forming the characteristic fibers of a meat-likeproduct. To make non-animal proteins palatable, texturization intofibrous meat analogs, for example, through extrusion processing has beenan accepted approach.

In more detail, extrusion processes are well known in the art andappropriate techniques can be determined by one of skill. “Extrusion” isa process used to create objects of a fixed cross-sectional profile. Amaterial is pushed or pulled through a die of the desired cross-section.High-moisture extrusion is known as wet extrusion. Extruders typicallycomprise an extruder barrel within which rotates a close-fitting screw.The screw is made up of screw elements, some of which are helical screwthreads to move material through the extruder barrel. Material isintroduced into the extruder barrel toward one end, moved along theextruder barrel by the action of the screw and is forced out of theextruder barrel through a nozzle or die at the other end. The rotatingscrew mixes and works the material in the barrel and compresses it toforce it through the die or nozzle. The degree of mixing and work towhich the material is subjected, the speed of movement of the materialthrough the extruder barrel and thus the residence time in the extruderbarrel and the pressure developed in the extruder barrel can becontrolled by the pitch of the screw thread elements, the speed ofrotation of the screw and the rate of introduction of material into theextruder barrel. The extruder barrel comprises multiple extruder barrelsections which are joined end to end. Multiple extruder barrel sectionsare required to carry out different processes involved in extrusion suchas conveying, kneading, mixing, devolatilizing, metering and the like.Each extruder barrel section comprises a liner which is press fit intoan extruder barrel casing, and heating and cooling elements are providedto regulate temperature of extruder barrel section within permissiblerange. The total length of an extrusion process can be defined by itsmodular extrusion barrel length. An extruder barrel is described by itsunit of diameter. A “cooling die” is cooling the extruded product to adesired temperature. Hot extrusion is used to thermomechanicallytransform raw materials in short time and high temperature conditionsunder pressure.

Steam-induced expansion, means melt expansion at the die exit due towater flashing off, hence leading to highly expanded products.Subsequent processing then determines the textural attributes ofextruded products such as crispness, crunchiness, hardness, etc. Theextruding can include, for example, melting and/or plasticization of theingredients, gelatinization of starch and denaturation of proteins. Theheat can be applied either through, for example, steam injection,external heating of the barrel, or mechanical energy. The material canbe pumped, shaped and expanded, which forms the porous and fibroustexture, and partially dehydrates the product. The shape and size of thefinal product can be varied by using different die configurations.Extruders can be used to make products with little expansion (such aspasta), moderate expansion (shaped breakfast cereal, meat substitutes,breading substitutes, modified starches, pet foods (soft, moist anddry)), or a great deal of expansion (puffed snacks, puffed curls andballs, etc.).

In some extruders, the material may be extruded by means of a ram or apiston. Other extruders use one or more screws. Variable pitch singlescrew extruders produce high product consistency by combining theingredients to produce a homogeneous mixture and pushing it out of themachine at a rate that is highly controllable. Twin screw extruderscontain two screws that are either co-current (the screws rotate in thesame direction) or are counter-current (the screws rotate in oppositedirections). Twin screw extruders can handle material with a wide rangeof moisture content and have greater control over the residence time andthe amount of shear to which the material is exposed.

The ingredients may be fed into the extruder via a feeder, such as, butnot limited to, a gravimetric or volumetric feeder. The type of feederused depends on the type of ingredient, and different feeders are usedfor batch versus continuous feed. The feeder also can direct theingredients into a preconditioner, if desired. The feed section of thescrew may have deep flights to accept the ingredients and move theingredients forward. The ingredients move into the compression sectionof the screw, which is heated, and has either shallower or more frequentflights, which compresses the ingredients and works them into continuousdough. The cooking section of the screw applies maximum heat, pressureand shear to the mixture in the barrel prior to the die. Within thescrew barrel, the mixture is heated and pressurized. When the mixtureemerges through the die, the reduction in pressure to atmosphericpressure generally causes the mixture to expand. If the moist doughwithin the barrel is heated over 100° C., the sudden reduction inpressure to atmospheric pressure causes the moisture to convert tosteam. The combination of sudden expansion and associated cooling yieldsa puffed, crisp product.

One suitable extrusion device is a double-barrel, twin screw extruder asdescribed, for example, in U.S. Pat. No. 4,600,311. Examples ofcommercially available double-barrel, twin screw extrusion apparatusinclude a CLEXTRAL Model BC-72 extruder manufactured by Clextral, Inc.(Tampa, Fla.); a WENGER Model TX-57 extruder manufactured by Wenger(Sabetha, Kans.); and a WENGER Model TX-52 extruder manufactured byWenger (Sabetha, Kans.). Other conventional extruders suitable for usein this disclosure are described, for example, in U.S. Pat. Nos.4,763,569, 4,118,164, and 3,117,006, which are hereby incorporated byreference herein.

The screws of a twin-screw extruder can rotate within the barrel in thesame (co-rotating) or opposite directions (counter-rotating). Rotationof the screws in the same direction is referred to as single flowwhereas rotation of the screws in opposite directions is referred to asdouble flow (counter-rotating). Typically, co-rotating twin screwextruders are used in the manufacturing of puffed snack products. Thespeed of the screw or screws of the extruder may vary depending on theparticular apparatus. However, the screw speed is typically from about200 to about 600 revolutions per minute (rpm). Generally, as the screwspeed increases, the density of the extrudates decreases. Particularly,the screw speed of the twin screw extruder may effect residence time ofthe dough in the extruder, the amount of shear generated, and the degreeof cooking of the dough; as the screw speed increases, residence timedecreases, and the amount of shear increases.

Among other things, conventional extruder systems generally act as aningredient mixer (e.g., to form the dough), mixture cooker, andcomposition (extrudate) former. Each of these functions can beaccomplished in the same cooking extruder. In some instances, however,it may be desirable to have at least two extruders arranged in a series.The extrusion apparatus also can include one or more heating zonesthrough which the dough is conveyed under mechanical pressure prior toexiting the extrusion apparatus through an extrusion die. The pressurewithin the extruder barrel is not narrowly critical. Typically, theextrusion mass is subjected to a pressure of at least about 400 psi(about 28 bar). The barrel pressure is dependent on numerous factorsincluding, for example, the extruder screw speed, feed rate of themixture to the barrel, feed rate of water to the barrel, and theviscosity of the dough within the barrel. Water can also be injectedinto the extruder barrel to further hydrate the dough. As an aid informing the dough the water may also act as a plasticizing agent. Watermay be introduced to the extruder barrel via one or more injection jetsin communication with a heating zone. Typically, water is injected at arate of from about 2 kg/hr to about 7 kg/hr. The mixture in the barreltypically contains from about 15% to about 30% (by weight) water. Therate of introduction of water to any of the heating zones is generallycontrolled to promote production of an extrudate having desiredcharacteristics. It has been observed that as the rate of introductionof water to the barrel decreases, the density of the extrudatedecreases. The dough in the extrusion apparatus is extruded through adie to produce an extrudate.

Extrusion conditions are generally such that the extrudate emerging fromthe extruder barrel typically has a moisture content of from about 5% toabout 20% (by weight extrudate). In one embodiment, the moisture contentof the extrudate is from about 5% (by weight extrudate) to about 15% (byweight extrudate). In another embodiment, the moisture content of theextrudate is about 10% (by weight extrudate). The moisture content isderived from water present in the mixture introduced to the extruder,moisture added during preconditioning and/or any water injected into theextruder barrel during processing. In an embodiment, high moistureextrusion e.g., extrusion at higher moisture contents (>40%), also knownas wet extrusion, can be used to create a fresh (non-dried) product.Generally, wet extrusion applications utilize twin screw extruders dueto their efficient conveying capabilities.

Upon release of pressure, the dough exits the extruder barrel throughthe die, superheated water present in the mass flashes off as steam,causing simultaneous expansion (i.e., puffing) of the material. Thelevel of expansion of the extrudate upon exiting of the mixture from theextruder in terms of the ratio of the cross-sectional area of extrudateto the cross-sectional area of die openings is generally less than about50:1. Typically, the ratio of the cross-sectional area of extrudate tothe cross-sectional area of die openings is from about 2:1 to about50:1. The extrudate may be cut after exiting the die. Suitable apparatusfor cutting the extrudate include flexible knives manufactured by Wenger(Sabetha, Kans.) and Clextral (Tampa, Fla.). Various die orifice designswill cause different expansions and geometric shapes of the extrudate.For example, depending on the geometric shape of the extrudate, thesliced or cut extrudate may be in the shape of a sheet, disc, pellet,rod, string, bar, and the like, or some other shape.

Other food compositions made using textured plant-based protein productsas disclosed herein include a burger, a patty, a sausage, a meatball,taco meat, or a frankfurter.

Such compositions of the invention (e.g., mixtures with myceliatedhigh-protein products and edible materials), using art-known methods,can be used to create a number of new food compositions, including,without limitation, dairy alternative products, ready to mix beveragesand beverage bases; extruded and extruded/puffed products; sheeted bakedgoods; meat analogs and extenders; bar products and granola products;baked goods and baking mixes; granola; and soups/soup bases. The methodsto prepare a food composition can include the additional, optional stepsof cooking, extruding, and/or puffing the food composition according tomethods known in the art to form the food compositions comprising themyceliated high protein food product of the invention.

The edible material can be, without limitation, a starch, a flour, agrain, a lipid, a colorant, a flavorant, an emulsifier, a sweetener, avitamin, a mineral, a spice, a fiber, a protein isolate/concentrate orpowder thereof, nutraceuticals, sterols, isoflavones, lignans,glucosamine, an herbal extract, xanthan, a gum, a hydrocolloid, astarch, a preservative, a legume product, a food particulate, andcombinations thereof. A food particulate can include cereal grains,cereal flakes, crisped rice, puffed rice, oats, crisped oats, granola,wheat cereals, protein nuggets, texturized plant protein ingredients,flavored nuggets, cookie pieces, cracker pieces, pretzel pieces, crisps,soy grits, nuts, fruit pieces, corn cereals, seeds, popcorn, yogurtpieces, and combinations of any thereof.

The edible materials can be added to the myceliated high proteinproducts of the invention using conventional processes to form theinventive food compositions, as described in more detail herein below.

In one embodiment, the food composition can include an alternative dairyproduct comprising a myceliated high protein food product according tothe invention. An alternative dairy product according to the inventionincludes, without limitation, products such as analog skimmed milk,analog whole milk, analog cream, analog fermented milk product, analogcheese, analog yogurt, analog butter, analog dairy spread, analog buttermilk, analog acidified milk drink, analog sour cream, analog ice cream,analog flavored milk drink, or an analog dessert product based on milkcomponents such as custard. Methods for producing alternative dairyproducts using alternative proteins, such as plant-based proteins asdisclosed herein including nuts (almond, cashew), seeds (hemp), legumes(pea), rice, and soy are known in the art. These known methods forproducing alternative dairy products using a plant-based protein can beadapted to use with a myceliated high protein food product usingart-known techniques.

An alternative dairy product according to the invention may additionallycomprise non-milk components, such as oil, protein, carbohydrates, andmixtures thereof. Dairy products may also comprise further additivessuch as enzymes, flavoring agents, microbial cultures, salts,thickeners, sweeteners, sugars, acids, fruit, fruit juices, any othercomponent known in the art as a component of, or additive to a dairyproduct, and mixtures thereof.

Milks. A myceliated high protein food product according to the inventionmay be used to create a myceliated high protein-based “milk” beverageproduced by using the myceliated high protein food product, optionally,by combining the product as a powder with oils and carbohydrates to forman emulsion, preferably a stable emulsion. Methods for creating veganprotein milks using soybeans as the protein source are known in the artand protein source may simply be substituted with myceliated highprotein food product protein. As a non-limiting example, a typicalunsweetened “milk” drink includes, per 243 ml serving, a total of 4 gcarbohydrates which can include 1 g of sugar, 4 g of fat or oil from anysource, and myceliated high protein food product solids sufficient toprovide between about 1-10 g of protein, the drink being in the form ofa stable emulsion of oil, water, and protein. The ratio of myceliatedhigh protein food product to the other ingredients can be varieddepending on the desired protein level of the drink and the desiredorganoleptic properties. Typically, the amount will vary between about0.1-10% g protein per mL beverage, or about 0.5 to 7%, 1% to 5% or about1.1-1.3%. The resulting slurry or purée may optionally be brought to aboil in order to e.g., improve its flavor, and to sterilize the product.Heating at or near the boiling point is continued for a period of time,15-20 minutes, followed by optional removal of insoluble residues bye.g., filtration.

In an example, the milk-based beverage can include 2.7 g myceliated highprotein food product per 240 mL serv, 4 g carbohydrates which caninclude 1 g of sugar, 4 g of fat or oil from any source.

Yogurt: myceliated high protein food product may be used to create amyceliated high protein food product-based “yogurt” beverage produced byusing myceliated high protein food product, optionally, by combiningmyceliated high protein food product with the other ingredients inpowder form. Methods for creating vegan yogurt using soybeans as theprotein source are known in the art and protein source may simply besubstituted with myceliated high protein food product protein, forexample, to create the yogurts of the invention. For example, myceliatedhigh protein food product can be used as 1.1% to about 7% (e.g., 10.7 g)myceliated high protein food product solids sufficient to providebetween about 1-10 g of protein per serving. Other ingredients in theyogurt can include, without limitation, as known in the art, nut milks(almond, cashew, for example), fats or oils (such as coconut cream,coconut oils), sugar, and thickening or gelling agents including,without limitation, agents such as locust bean gums, pectin, and thelike. The composition, in some embodiments, will contain no less than2.5% fat from a plant source, such as, without limitation, almond,cashew, and/or coconut and no less than 3.5% protein. Frozen yogurtswill have similar compositions.

In an example, the yogurt can include 68.7% by weight of an almond milk,21.9% of a cashew milk, 3.35% of coconut cream, 4.75% of myceliated highprotein food product, 1.18% of dextrose, 0.05% of locust bean gum, 0.05%of pectin, and 0.02% of live bacterial cultures customary for yogurtpreparations, such as mixtures of lactic acid producing bacteriaLactobacillus bulgaricus and Streptococcus thermophilus. For a frozendessert, example amounts of myceliated high protein food product caninclude about 4 g myceliated high protein food product per 79 g serving(cashew) or 6.67 g myceliated high protein food product per 85 gserving.

Ice Cream: myceliated high protein food product may be used to create amyceliated high protein food product-based “ice cream” beverage producedby using myceliated high protein food product, optionally, by combiningmyceliated high protein food product with the other ingredients inpowdered form. Methods for creating vegan ice cream using soybeans asthe protein source are known in the art and protein source may simply besubstituted with myceliated high protein food product protein, forexample, to create the ice creams of the invention. For example,myceliated high protein food product can be used as 1.1% to 7% (10.7 g)myceliated high protein food product solids sufficient to providebetween about 1-10 g of protein per serving. Other ingredients in theice cream can include, without limitation, as known in the art, creams,fats or oils (such as coconut cream, coconut oil), sugar, and thickeningor gelling agents including, without limitation, agents such as locustbean gum, pectin, emulsifiers such as lecithin, and the like. Thecomposition, in some embodiments, will contain no less than 10% fat froma plant source, such as, without limitation, almond, cashew, and/orcoconut and no less than 3.5% protein and no less than 35% total solids.

In an example, the ice cream can include 45.5% by weight of water, 32%of coconut cream (34.7% fat), 4.5% of myceliated high protein foodproduct 17% of sugar, 0.6% of a gum, 0.2% of lecithin, 0.2% of sea salt.

The present invention can also include beverages and beverage basescomprising a myceliated high protein food product according to theinvention which can be used as non-dairy-based meal replacementbeverages. A myceliated high protein food product according to theinvention may be used to prepare a meal replacement beverage that isoptionally non-dairy-based. Methods for creating vegan meal replacementbeverages using soybeans as the protein source are known in the art andprotein source may simply be substituted with myceliated high proteinfood product protein of the invention, for example. For example, atypical meal replacement drink would include, per 243 ml serving, atotal of 4 g carbohydrates which can include 1 g of sugar, 4 g of fat oroil from any source, and myceliated high protein food product solidssufficient to provide between about 2-30 g of protein. The ratio ofmyceliated high protein food product can be varied depending on thedesired protein level of the drink and the desired organolepticproperties. Typically, the amount will vary between about 0.1-15% gprotein per mL beverage, or about 0.5 to 7%, 1% to 5% or about 1.1-1.3%.The resulting slurry or purée may optionally be brought to a boil inorder to e.g., improve its flavor, and to sterilize the product. Heatingat or near the boiling point is continued for a period of time, 15-20minutes, followed by optional removal of insoluble residues by e.g.,filtration. A ready to mix beverage powder can include 32.7 g ofmyceliated high protein food product per 35 g serving. Examples ofproducts include protein shakes and smoothies, and dietary andnutritional beverages including meal replacement beverages andsmoothies.

In an exemplary formulation, a non-dairy-based meal replacement beveragecan have about 20 g of the myceliated high protein food product per 243g serving.

The present invention can also include extruded and/or puffed productsand/or cooked products comprising a myceliated high protein food productof the invention. Extruded and/or puffed ready-to-eat breakfast cerealsand snacks such as crisps or scoops and pasta noodles are known in theart. Extrusion processes are well known in the art and appropriatetechniques can be determined by one of skill. “Extrusion” is a processused to create objects of a fixed cross-sectional profile. A material ispushed or pulled through a die of the desired cross-section. The twomain advantages of this process over other manufacturing processes areits ability to create very complex cross-sections, and to prepareproducts that are brittle, because the material only encounterscompressive and shear stresses. High-moisture extrusion is known as wetextrusion. Extruders typically comprise an extruder barrel within whichrotates a close-fitting screw. The screw is made up of screw elements,some of which are helical screw threads to move material through theextruder barrel. Material is introduced into the extruder barrel towardone end, moved along the extruder barrel by the action of the screw andis forced out of the extruder barrel through a nozzle or die at theother end. The rotating screw mixes and works the material in the barreland compresses it to force it through the die or nozzle. The degree ofmixing and work to which the material is subjected, the speed ofmovement of the material through the extruder barrel and thus theresidence time in the extruder barrel and the pressure developed in theextruder barrel can be controlled by the pitch of the screw threadelements, the speed of rotation of the screw and the rate ofintroduction of material into the extruder barrel. The extruder barrelcomprises multiple extruder barrel sections which are joined end to end.Multiple extruder barrel sections are required to carry out differentprocesses involved in extrusion such as conveying, kneading, mixing,devolatilizing, metering and the like. Each extruder barrel sectioncomprises a liner which is press fit into an extruder barrel casing, andheating and cooling elements are provided to regulate temperature ofextruder barrel section within permissible range. The total length of anextrusion process can be defined by its modular extrusion barrel length.An extruder barrel is described by its unit of diameter. A “cooling die”is cooling the extruded product to a desired temperature.

For example, cold extrusion is used to gently mix and shape dough,without direct heating or cooking within the extruder. In foodprocessing, it is used mainly for producing pasta and dough. Theseproducts can then be subsequently processed: dried, baked,vacuum-packed, frozen, etc.

Hot extrusion is used to thermomechanically transform raw materials inshort time and high temperature conditions under pressure. In foodprocessing, it is used mainly to cook biopolymer-based raw materials toproduce textured food and feed products, such as ready-to-eat breakfastcereals, snacks (savory and sweet), pet foods, feed pellets, etc. Theextruding can include, for example, melting and/or plasticization of theingredients, gelatinization of starch and denaturation of proteins. Theheat can be applied either through, for example, steam injection,external heating of the barrel, or mechanical energy. The material canbe pumped, shaped and expanded, which forms the porous and fibroustexture, and partially dehydrates the product. The shape and size of thefinal product can be varied by using different die configurations.Extruders can be used to make products with little expansion (such aspasta), moderate expansion (shaped breakfast cereal, meat substitutes,breading substitutes, modified starches, pet foods (soft, moist anddry)), or a great deal of expansion (puffed snacks, puffed curls andballs, etc.).

The myceliated high protein food product of the invention may be used informulating foods made by extrusion and/or puffing and/or cookingprocesses, such as ready to eat breakfast cereals and snack foods. Thesematerials are formulated primarily with cereal grains and may containflours from one or more cereal grains. The cereal grains utilized, suchas corn, wheat, rice, barley, and the like, have a high starch contentbut relatively little protein. A cereal having more protein content,therefore, is desirable from a nutritional standpoint. The compositionof the present invention contain flour from at least one cereal grain,preferably selected from corn and/or rice, or alternatively, wheat, rye,oats, barley, and mixtures thereof. The cereal grains used in thepresent invention are commercially available, and may be whole graincereals, but more preferably are processed from crops according toconventional processes for forming refined cereal grains. The term“refined cereal grain” as used herein also includes derivatives ofcereal grains such as starches, modified starches, flours, otherderivatives of cereal grains commonly used in the art to form cereals,and any combination of such materials with other cereal grains. Arefined corn for example, is formed from U.S. No. 1 or No. 2 yellow dentcorn by dry milling the corn to separate the endosperm from the germ andbran, and forming corn meal, corn grits, or corn flour from theendosperm. Refined wheat grain may be formed according to commercialmilling practices from hard or soft wheat varieties, red or white wheatvarieties, and may be a wheat flour containing little or no wheat bran,a wheat bran, or a milled wheat product containing flour, bran, and germ(whole wheat flour). Refined rye is preferably a rye flour which isformed according to commercial milling practices. Refined rice may beheads, second heads, or brewers rice which is formed by conventionalpractices for dehulling rough rice and pearling the dehulled rice, andpreferably rough grinding the pearled and dehulled rice into a riceflour. Oats are refined by conventional practices into oat meal bydehulling and cleaning the oats to form oat groats and milling the oatgroats to form oat meal or oat flour. The refined oats may also bedefatted. Barley is refined according to conventional practices intobarley flakes or barley grits by dehulling and cleaning the barley toform clean barley which is pearled and flaked or ground to form thebarley flakes or barley grits.

The breakfast cereal and snack materials can obtain the desired flakestructure by a process known as puffing. Basically, a cereal is puffedby causing trapped moisture in the flake to change very rapidly from theliquid state to the vapor phase. Rapid heating or a rapid decrease inpressure are the methods commonly used throughout the industry. Gunpuffing is an example of the principle of a rapid decrease in pressure.In this process the cereal flakes are first heated under high pressureand then the pressure is rapidly released to achieve the puffing effect.The process disclosed in U.S. Pat. No. 3,253,533 is an example of arapid heating puffing method.

To achieve the optimum puffing, care must be taken in regard to theinitial moisture content of the unpuffed flake. The specific moisturecontent that is best is dependent on the particular type of puffingprocess being utilized. For instance, a moisture content of 12 to 14percent is best for gun puffing while to 12 percent is best for puffingby a process that rapidly heats the flake. The optimum moisture contentfor any one puffing technique can routinely be determinedexperimentally. Additional processing steps can be utilized if it is sodesired. For instance a toasting operation can be used after the puffingstep if it is desired to change the color of the flake to a more desiredrich golden brown. Frequently, a slight toasting step also brings out apleasant toasted flavor note.

The food product produced using the methods described herein can be inthe form of crunchy curls, puffs, chips, crisps, crackers, wafers, flatbreads, biscuits, crisp breads, protein inclusions, cones, cookies,flaked products, fortune cookies, etc. The food product can also be inthe form of pasta, such as dry pasta or a ready-to-eat pasta. Theproduct can be used as or in a snack food, cereal, or can be used as aningredient in other foods such as a nutritional bar, breakfast bar,breakfast cereal, or candy. In a pasta, the myceliated high protein foodproduct may be, in a non-limiting example, be used in levels of about 10g per 58 g serving (17%).

Baked Goods.

Food compositions of the invention also include bakery products andbaking mixes comprising myceliated high protein food products accordingto the invention according to known methods. The term “bakery product”includes, but is not limited to leavened or unleavened, traditionallyflour-based products such as white pan and whole wheat breads (includingsponge and dough bread), cakes, pretzels, muffins, donuts, brownies,cookies, pancakes, biscuits, rolls, crackers, pie crusts, pizza crusts,hamburger buns, pita bread, and tortillas.

In accordance with embodiments of the invention, leavening agents may beincluded in dough to produce products, which require a rising, such ascrackers and breads. Exemplary leavening agents include yeast, bakingpowder, eggs, and other commercially available leavening agents.Preferably, leavening agents will comprise less than about 5%, byweight, of the dry ingredients.

Dough in accordance with embodiments of the invention may also includegums such as xanthum, guar, agar, and other commercially availablehydrocolloids typically used for dough binding and conditioning.Additionally, food grade oils can be used to improve sheeting, texture,browning, and taste. Exemplary oils include soybean oil, canola oil,corn oil, and other commercially available oils. Lecithin may also beadded to improve emulsification, water binding, and dough release.

In an embodiment, the amount of myceliated high protein food product inthe bakery products or bakery mixes is in the range of at least 2 to 7grams per 50 gram serving, or 5 or 6 grams per serving. A method ofproducing a food composition of the invention includes forming acohesive dough by measuring and mixing the dry ingredients usingstandard mixing equipment.

Bread, rolls, bagels, and English muffins according to the invention mayhave between about 4.8% to about 7% (2.7 g) myceliated high protein foodproduct of the invention per 40 g serving (adding 2 g protein for highprotein bread formulation.)

Bars and Granolas

The present invention also includes food compositions such as granolacereals, and bar products, including such as granola bars, nutritionbars, energy bars, sheet and cut bars, extruded bars, baked bars, andcombinations thereof.

The baked food compositions and bar compositions are generally formeddependent on the desired end product. The baked food compositions andbar compositions are produced according to standard industry recipes,substituting in a myceliated high-protein food product of the presentinvention for at least some of the called-for protein ingredients.

For the extruded compositions, protein fortification may be accomplishedby supplementing the bar with edible proteins from at least one highprotein content source, as known in the art, and including themyceliated food product of the present invention, either alone or ascombinations with other proteins Based upon the weight of the extrudate,or core, a suitable amount of the at least one high protein contentsource is about 20% to about 30% by weight. The protein content shouldbe at least about 15% by weight, based upon the weight of the finalproduct.

In the present invention, a liquid sweet ingredient, such as corn syrup,preferably high fructose corn syrup, is used as a carbohydrate contentsource. In one embodiment, the liquid sweet ingredient provides a moistchewy texture to the bar, provides sweetness, and serves to distributethe dry ingredients. The liquid sweet ingredient can include, withoutlimitation, corn syrup, high fructose corn syrup, honey, tapioca syrup,among others as known in the art. Additionally, the liquid sweetingredient, in combination with other binders known in the art, can beuseful to bind the other ingredients, such as the protein content andother carbohydrate content sources together. Suitable amounts of theliquid sweet ingredient are about 25% to about 30% by weight, based uponthe weight of the extrudate. At least one other carbohydrate contentsource may be optionally included in the bar of the present invention.Exemplary of suitable carbohydrate content sources for providing acaloric distribution within the above ranges are sugars, such asfructose granules, brown sugar, sucrose, and mixtures thereof, andcereal grains such as rice, oats, corn, and mixtures thereof.Preferably, the snack contains at least one sugar and at least onecarbohydrate. Based upon the weight of the core, suitable amounts ofthese ingredients are about 3% to about 10% by weight of at least onesugar, and about 12% to about 18% by weight of at least one cerealgrain. The bar also optionally comprises a fat. Suitable sources of fatsinclude those known in the art to be suitable for bar-type products andinclude milk, chocolate, and coconut oils, creams, and butters; nutbutters such as peanut butter, and an oil such as vegetable oil. Also, aliquid wetting agent may be present in the composition, to facilitatemixing and binding of the dry ingredients to enhance moistness andchewiness of the snack. Exemplary of such wetting agents are molasses,honey, and vegetable oils, and mixtures thereof. A suitable amount ofthe at least one wetting agent is about 2% to 5% by weight. Suitableamounts of the flavoring ingredients range up to about 3% by weight.Also it is known in the art that carbohydrate content sources, useful inthe present invention, may also be substantial sources of proteinsand/or fats. For example, peanut flour, oats, and wheat germ eachprovide substantial amounts of proteins, carbohydrates, and fats.Dietary fiber can be included in the bar. Suitable amounts are about 3%to about 8%, preferably about 5% by weight fiber, based upon the weightof the final product. Suitable sources of dietary fiber are rolled oatsand brans. The bar may be topped with conventional toppings, such asgranola, crushed nuts, and the like, to enhance flavor and visualappeal. Suitable topping amounts are about 2% to 3% by weight of thefinal product.

In one embodiment, the nutritional snacks of the present invention aremade by first mixing the liquid ingredients and the optional wettingagent. Next, the minor dry components are added to the mixed liquids.The minor dry components include ingredients such as, for example,minerals and vitamins, preferably premixed, and optional salt. The majordry ingredients can then admixed with the mixed liquids and minor dryingredients to form a substantially homogeneous mixture. The major dryingredients include e.g., sugars and cereal grains. The major dryingredients also include the high protein content sources including themyceliated high protein food product of the invention. The flavoringingredients, such as cocoa or coconut, can be added with the minor dryingredients or with the major dry ingredients. All mixing can be in thesame mixer or blender. Suitable mixing and blending equipment includeconventional vertical and horizontal type mixers and blenders.

The mixed ingredients can be transferred via conveyor belts and hoppers,for example, to a conventional bar extruder, having opposing rollerswhich force the mixture through a die to form the extrudate or core. Theextrusion is performed at about room temperature. No cooking or heatingduring or after extrusion is necessary nor desirable. The preferredextruded shape is a rectangular bar, but other shaped bars, known in thesnack bar art, such as cylindrical, and semicylindrical shaped bars canbe made using appropriate extruder dies.

In accordance with the present invention, the granola cereals and barproducts, the dry ingredients can include a food particulate. A foodparticulate may include, without limitation, any edible foodparticulate. Such particulates can include flours, meals, cereal grains,cereal flakes, crisped rice, puffed rice, oats, crisped oats, granola,wheat cereals, protein nuggets, textured soy flour, textured soy proteinconcentrate, texturized protein ingredients such as those disclosedherein, flavored nuggets, cookie pieces, cracker pieces, pretzel pieces,crisps, soy grits, nuts, fruit pieces, vegetable pieces, corn cereals,seeds, popcorn, yogurt pieces, and combinations of any thereof.

For example, for grain-based bars, an appropriate amount of myceliatedhigh protein food product includes from between about 20% to about 33.3%(20 g) myceliated high protein food product per 60 g serving (forexample, 15 g protein in a high protein bar). Where the bar contains afruit and/or vegetable, an appropriate amount of myceliated high proteinfood product includes can include about 20% (8 g) myceliated highprotein food product per 45 g serving (adding 6 g to a total of 8 g in ahigh protein type bar.)

After extrusion, the product may be dried. The final product will have amoisture content of from about 1% to about 8%, depending on the desiredcharacteristics of the finished product.

In one embodiment, an extruded nutritional protein bars may include21.33 g/60 g of myceliated high protein food product of the presentinvention, with the balance including carbohydrate, nuts, oils, withproportions determined by conventional processes known in the art.

Food compositions of the present invention also include smoothies andsmoothie bases, and juices, and soups and soup bases, fats and oils. Forexample, salad dressings can include about 8 g myceliated high proteinfood product of the invention per 30 g serving; a fruit juice, fruitflavored drink, fruit nectar may include about 1% by weight ofmyceliated high protein product of the invention. A vegetable juice suchas a tomato juice can include between about 2.5% to about 20% (8 g)myceliated high protein food product of the invention per 240 mLserving. A smoothie may contain between about 3.5% to 20% by weight orbetween 9 and 20 g of myceliated high protein product of the invention,for example about 40 g per 450 mL serving.

For a soup or soup base (mix), prepared soups, dry soup mixes, andcondensed soups, a myceliated high protein food product may be added inan amount of between 0.96%-˜3.3% by weight (8 g) per 242 g serving. Fora confectionary, such as a chocolate dessert (peanut butter cup), amyceliated food product of the invention may include about 2.67 g per 40g serving.

Reaction Flavors

The Maillard reaction is a chemical reaction between amino acids andreducing sugars that gives browned food its distinctive flavor. Searedsteaks, pan-fried dumplings, cookies and other kinds of biscuits,breads, toasted marshmallows, and many other foods undergo thisreaction. The reaction is a form of non-enzymatic browning whichtypically proceeds rapidly from around 140 to 165° C. (280 to 330° F.).Many recipes call for an oven temperature high enough to ensure that aMaillard reaction occurs. At higher temperatures, caramelization andsubsequently pyrolysis become more pronounced. In a Maillard reaction,the reactive carbonyl group of the sugar reacts with the nucleophilicamino group of the amino acid and forms a complex mixture of poorlycharacterized molecules responsible for a range of aromas and flavors.This process is accelerated in an alkaline environment (e.g., lyeapplied to darken pretzels; see lye roll), as the amino groups(RNH3+→RNH2) are deprotonated, hence have an increased nucleophilicity.The type of the amino acid determines the resulting flavor. Thisreaction is the basis for many of the flavoring industry's recipes. Athigh temperatures, a potential carcinogen called acrylamide can beformed.

The Strecker degradation (SD) plays several roles in the formation offlavor compounds in processed foods. Primarily, it is the major pathwayfor conversion of amino acids into structurally related aldehydes ofsignificant flavor value. Also, the SD provides a relatively low energyroute for mobilizing amino acids' nitrogen and sulfur to form ammonia,hydrogen sulfide and many flavor-significant S/N/O-containingheterocyclic compounds. And finally, the SD provides a reductionmechanism for conversion of dicarbonyls into acyloins thereby openingthe door to still more diverse flavor compound formation.

In one embodiment, the present invention includes a method to prepare areaction flavor composition. In this embodiment, the edible materialcomprises providing at least one reaction flavor component capable offacilitating Maillard and/or Strecker reactions. In another step, themethod includes mixing the myceliated high protein food product and thereaction flavor component. In yet another step, the method includesprocessing the mixture to form the reaction flavor composition.

The present invention, in one embodiment, is directed to a reactionflavor. A “reaction flavor” is an art-recognized term that describes aflavor composition that is capable of providing, modifying or improvingthe flavor of foods and beverages. A “reaction flavor” is one thatoccurs by way of chemical reactions upon addition of sugars, amino acidsand heat, such as Maillard reactions and Strecker reactions. TheMaillard reaction is a chemical reaction between amino acids andreducing sugars that gives browned food its distinctive flavor. Searedsteaks, pan-fried dumplings, cookies and other kinds of biscuits,breads, toasted marshmallows, and many other foods undergo thisreaction. The reaction is a form of non-enzymatic browning whichtypically proceeds rapidly from around 140 to 165° C. (280 to 330° F.).Many recipes call for an oven temperature high enough to ensure that aMaillard reaction occurs. At higher temperatures, caramelization andsubsequently pyrolysis become more pronounced. In a Maillard reaction,the reactive carbonyl group of the sugar reacts with the nucleophilicamino group of the amino acid and forms a complex mixture of poorlycharacterized molecules responsible for a range of aromas and flavors.This process is accelerated in an alkaline environment (e.g., lyeapplied to darken pretzels; see lye roll), as the amino groups(RNH₃+→RNH₂) are deprotonated, hence have an increased nucleophilicity.The type of the amino acid determines the resulting flavor. Thisreaction is the basis for many of the flavoring industry's recipes. Athigh temperatures, a potential carcinogen called acrylamide can beformed.

A Maillard reaction relies mainly on sugars and amino acids but it canalso contain other ingredients including: autolysed yeast extracts(AYE), hydrolysed vegetable proteins (HVP), gelatin (protein source),vegetable extracts (i.e. onion powder), enzyme treated proteins, meatfats or extracts and acids or bases to adjust the pH of the reaction.The reaction is aqueous with an adjusted pH at specific temperatures(typically 100° C.) for a specified amount of time (typically 15 mins)to produce a variety of flavors. Typical flavors yielded are chicken,pork, beef, caramel, and chocolate. However, a wide variety of nuancesand intensities can be achieved by adjusting the ingredients, thetemperature and/or the pH of the reaction. The main advantage of thereaction flavor is that it can produce characteristic meat, burnt,roasted, caramellic, or chocolate profiles desired by the food industry,which are not typically achievable by using compounding of flavoringredients.

Both mono as well as disaccharides can take part in the Maillardreaction. Generally speaking aldoses are more reactive than ketoses andpentoses more than hexoses or disaccharides, and so whereas the type ofsugar strongly influences the amount of flavoring compounds generated,the amino acid involved in the reaction largely determines the nature ofthe flavor formed. For example, the inclusion of pure methionine inMaillard reaction systems often leads to vegetable or stewed notes, purecysteine leads to meat-like flavors, pure proline, hydroxy proline andleucine to bakery aromas (R. F. Hurrell, Food Flavours, Part A:Introduction, Elsevier Scientific Publishing Company, Eds.: I. D. Mortonand A. J. Macleod). Since these results have been obtained using pureamino acids rather than mixtures of several amino acids, as occur infood ingredients, it is evident that the outcome represents only a grosssimplification of the natural situation. Likewise, the sugars thatnaturally occur in food will have an impact, and further complicate andaffect the development of taste and aroma.

The Strecker degradation reaction is instrumental in the creation of thebrown pigment as well as a myriad of volatile aromatics. It falls underthe umbrella of, and requires compounds created by, the MaillardReactions. Strecker degradation produces Strecker aldehydes and2-aminocarbonyl compounds, both are critical intermediates in thegeneration of aromas during Maillard reaction, however, they can also beformed independently of the pathways established for Streckerdegradation. Strecker aldehyde can be formed directly either from freeamino acids or from Amadori products. Several pathways have beenproposed in the literature for the mechanism of this transformation. Onthe other hand, Amadori or Heyns rearrangements of ammonia with reducingsugars can also generate 2-aminocarbonyl compounds without the formationof Strecker aldehyde. In addition, isomerization of the imine bond ofthe Schiff base formed between a reducing sugar and an amino acid, caninitiate a transamination reaction and convert the amino acid into thecorresponding α-keto acid and the sugar into its α-amino alcoholderivative. The reverse of this reaction has been documented to produceAmadori products. The α-keto acids can either decarboxylate to produceStrecker aldehydes or undergo Strecker degradation (as a α-dicarbonylcompound) with amino acids to also produce Strecker aldehydes. Keyintermediates include α-dicarbonyl, α-hydroxycarbonyl, 2-amino carbonylsand 2-(amino acid)-carbonyl compounds during the Maillard reaction.

A reaction flavor, in an embodiment, will consist of a complexmulti-component blend of both volatile and non-volatile reactionproducts, as well as any unreacted starting materials. Importantreaction parameters can include precursor ingredient chemistry, reactiontime and temperature, moisture, pressure, pH and the like. An importantprocess parameter is viscosity control which will affect stirring,blending, pumping and the like, and therefore in many cases, it isconventional to form reaction flavors in aqueous slurries. Usingconventional techniques, after cooking a slurry to create a reactionflavor, the slurry is dehydrated in a second step by spray drying orvacuum oven drying. Other methods known in the art include microwaveprocessing, such as, for example, as disclosed in WO 2018/083224,published 11 May 2018, which is incorporated herein by reference in itsentirety.

In one embodiment of the invention, the precursor material for thereaction flavor is a myceliated high-protein food product as disclosedherein and as made by processes disclosed herein. To the myceliated highprotein food product as disclosed herein, a number of precursorcompounds can be added, as known in the art, which can be varied in amanner known by a skilled flavorist, depending on the particularreaction flavor that is desired to create. Precursor compounds that canbe added to the myceliated high protein food product include aminoacids/amine sources, reducing sugars, as well as lipids or fats, spicesand additional protein sources, such as hydrolyzed vegetable proteins(HVPs) or yeast autolysates.

In addition to these materials, the slurry can contain other materialsthat can modify taste or flavor, as known in the art, including sulfursources, meat powers, powdered broths or stocks, and others.

In addition to the myceliated high protein food product as disclosedherein, additional amino acid/amine sources may be added, such as, forexample, cysteine, methionine, alanine, glycine, lysine, arginine,histidine, tryptophan, proline, valine, glutamic acid, glutamine,aspartic acid, glutathione, other sulfur-containing peptides, HVP(groundnut, soybean wheat/maize gluten), other hydrolyzed proteins(e.g., from milk, egg, fish, blood, liver, bone, collagen), yeastextract, autolyzed yeast, meat extract, taurine and pyrrolidonecarboxylic acid.

Reducing sugars are those that either have an aldehyde group or arecapable of forming one in solution. The aldehyde group allows the sugarto act as a reducing agent in the Maillard reaction. Cyclic hemiacetalforms of aldoses can open to provide an aldehyde and certain ketoses canundergo tautomerization to become aldoses. Examples of reducing sugarsinclude glucose, fructose, xylose, glyceraldehyde, galactose, lactose,arabinose, maltose, glucose polymers such as starch, hydrolyzed starch,and starch-derivatives such as glucose syrup, maltodextrin, and dextrin.Reaction flavor components can include glucose, ribose, fructose, datesyrup, high fructose corn syrup, malted barley, agave syrup, tapiocasyrup, and brown rice syrup, calcium carbonate, ascorbic acid, sodiumascorbate, calcium ascorbate, and/or potassium ascorbate, andcombinations thereof.

Sulfur sources can include hydrogen sulfide, cysteine, cystine,methionine, glutathione, thiamine, inorganic sulfides, organic thiolsand sulfides, 2-mercaptoethanol derivatives, vegetable extracts,fermented vegetable juices, yeast extract, autolyzed yeast, egg proteinand meat extract.

The ratio between an amino acid and reducing sugar can vary within widelimits, as known in the art. In one embodiment, a typical ratio of aminoacid to carbohydrate is 1:5 but this can vary significantly depending onthe effect to be achieved. In an embodiment, the reaction flavor will bethe product of a slurry containing up to 30 wt % water, up to 70% wt %of protein, 6 wt % of reducing sugar, up to 4 wt % lipids, up to 20 wt %carrier.

The pH of the slurry can be adjusted in the range of 0.5 to 8, moreparticularly 4 to 8. Any food grade acids or bases can be used, forexample, lactic acid, phosphoric acid, acetic acid, citric acid, malicacid, tartaric acid, oxalic acid, tannic acid, and combinations thereof;and examples of bases include sodium hydroxide, sodium carbonate,potassium bicarbonate, and sodium acetate.

The reaction flavor solid compositions of the present invention mayrepresent a complete flavor composition that may be blended with a foodor beverage to impart flavor thereto, or modify or improve the flavorthereof. Alternatively, the reaction flavor solid composition may formonly a part of a complete flavor composition, and the reaction flavorsolid composition can be mixed with other flavor ingredients to form thecomplete flavor composition.

A skilled flavorist will be able to mix a reaction flavor solidcomposition of the present invention with other known ingredientsemployed in flavor compositions to develop a wide variety of completeflavor compositions to satisfy the requirements of the food and beverageindustry. Those other known ingredients useful in complete flavorcompositions may be added to the slurry before the formation of thereaction flavor solid composition, or they may be blended with areaction flavor solid composition after it is formed, or both.

A complete flavor composition may comprise a reaction flavor solidcomposition as described herein; aroma volatiles and other flavoringredients generally known in the art; and other synergists orenhancers, such as fats or fatty acids, or their sources, herbs, spicesand the like; pH regulators; inorganic salts; taste masking agents,vitamins; dyes; colorants; pigments, and the like.

Other ingredients include aldehyde and ketone sources, includingacetaldehyde, propanal, butanal, methylpropanal, C3 to C5 alkanals, HVP,alpha diketones and sources thereof, including butanedione,pentane-2,3-dione, pyruvaldehyde, pyruvic acid, glyceraldehyde, glyoxal,dihydroxyacetone, alpha-ketobutyric acid,heptane-3,4-dione-2,5-diacetate, HMFone, HDFone, and relatedderivatives, ascorbic acid, 5-ketogluconic acid, cyclotene, maltol,lactic acid, glycolic acid, malic acid, tartaric acid, and proteinhydrolysates.

Examples of flavor enhancers and their sources include MSG, IMP, GMP,yeast extract, autolyzed yeast, HVP,2-furfuryl-thioinosine-5′-phsophate, 2-allyloxyinosine-5′-phosphate,2-(lower alkoxy) inosine-5′-phosphate, 2-benzylthioinosine-5′-phosphate,4-glucosylgluconic acid, and cyclotene.

Examples of fats include fats of beef, chicken, coconut, othertriglycerides, fatty acids, and their esters. Examples of inorganicsalts include chlorides and phosphates.

Depending upon the flavor profile that a skilled flavorist is trying toachieve, a complete flavor composition might additionally contain one ormore of the following ingredients: acetaldehyde (apple), dimethylsulfide, ethyl acetate, ethyl propionate, methyl butyrate, and ethylbutyrate; flavor oils containing volatile aldehydes or esters include,e.g., cinnamyl acetate, cinnamaldehyde, citral, diethylacetal,dihydrocarvyl acetate, eugenyl formate, and p-methylanisole. Furtherexamples of volatile compounds that may be present in the flavor oilsinclude: benzaldehyde (cherry, almond); cinnamic aldehyde (cinnamon);citral, i.e., alpha citral (lemon, lime); neral, i.e., beta-citral(lemon, lime); decanal (orange, lemon); ethyl vanillin (vanilla, cream);heliotropine, i.e., piperonal (vanilla, cream); vanillin (vanilla,cream); alpha-amyl cinnamaldehyde (spicy fruity flavors); butyraldehyde(butter, cheese); valeraldehyde (butter, cheese); citronellal (modifies,many types); decanal (citrus fruits); aldehyde C-8 (citrus fruits);aldehyde C-9 (citrus fruits); aldehyde C-1 (citrus fruits); 2-ethylbutyraldehyde (berry fruits); hexenal, i.e., trans-2 (berry fruits);tolyl aldehyde (cherry, almond); veratraldehyde (vanilla);2,6-dimethyl-5-heptenal, i.e., melonal (melon); 2-6-dimethyloctanal(green fruit); and 2-dodecenal (citrus, mandarin); cherry; or grape andmixtures thereof; spice oleoresins derived from allspice, basil,capsicum, cinnamon, cloves, cumin, dill, garlic, marjoram, nutmeg,paprika, black pepper, rosemary, and turmeric, essential oils, aniseoil, caraway oil, clove oil, eucalyptus oil, fennel oil, garlic oil,ginger oil, peppermint oil, onion oil, pepper oil, rosemary oil,spearmint oil, citrus oil, orange oil, lemon oil, bitter orange oil,tangerine oil, alliaceous flavours, garlic, leek, chive, and onion,botanical extracts, arnica flower extract, chamomile flower extract,hops extract, marigold extract, botanical flavour extracts, blackberry,chicory root, cocoa, coffee, kola, licorice root, rose hips,sarsaparilla root, sassafras bark, tamarind and vanilla extracts,protein hydrolysates, hydrolyzed vegetable proteins, meat proteinhydrolyzes, milk protein hydrolyzates and compounded flavours bothnatural and artificial including those disclosed in S. Heath, SourceBook of Flavors, Avi Publishing Co., Westport Conn., 1981, pages149-277; valerian oil; 3,4-dimeth-oxyphenol; amyl acetate; amylcinnamate, g-butyryl lactone; furfural; trimethyl pyrazine; phenylacetic acid; isovaleraldehyde; ethyl maltol; ethyl vanillin; ethylvalerate; ethyl butyrate; cocoa extract; coffee extract; peppermint oil;spearmint oil; clove oil; anethol; cardamom oil; wintergreen oil;cinnamic aldehyde; ethyl-2-methyl valerate; g-hexenyl lactone;2,4-decadienal; 2,4-heptadienal; methyl thiazole alcohol(4-methyl-5-p-hydroxyethyl thiazole); 2-methyl butanethiol;4-mercapto-2-butanone; 3-mercapto-2-pentanone; 1-mercapto-2-propane;benzaldehyde; furfural; furfuryl alcohol; 2-mercapto propionic acid;alkyl pyrazine; methyl pyrazine; 2-ethyl-3-methyl pyrazine; tetramethylpyrazine; polysulfides; dipropyl disulfide; methyl benzyl disulfide;alkyl thiophene; 2,3-dimethyl thiophene; 5-methyl furfural; acetylfuran; 2,4-decadienal; guiacol; phenyl acetaldehyde; b-decalactone;D-limonene; acetoin; amyl acetate; maltol; ethyl butyrate; levulinicacid; piperonal; ethyl acetate; n-octanal; npentanal; n-hexanal;diacetyl; monosodium glutamate; monopotassium glutamate;sulfur-containing amino acids, e.g., cysteine; hydrolyzed vegetableprotein; 2-methylfuran-3-thiol; 2-methyldihydrofuran-3-thiol;2,5-dimethylfuran-3-thiol; hydrolyzed fish protein; tetramethylpyrazine; propylpropenyl disulfide; propylpropenyl trisulfide; diallyldisulfide; diallyl trisulfide; dipropenyl disulfide; dipropenyltrisulfide; 4-methyl-2-[(methylthio)-ethyl]-1,3-dithiolane;4,5-dimethyl-2-(methylthiomethyl)-1,3-dithiolane; and4-methyl-2-(methylthiomethyl)-1,3-dithiolane.

Complete flavor compositions may contain taste masking agents. Tastemasking agents are substances for masking one or more unpleasant tastesensations, in particular a bitter, astringent and/or metallic tastesensation or aftertaste, which substances can be a constituent of theproducts according to the invention. Examples include dihydrochalcones,nucleotides, sodium salts, hydroxyflavanones and the like.

Complete flavor compositions may contain taste sensates. Taste sensatesinclude hot tasting, salivation-inducing substances, substances causinga warm or tingling feeling, and cooling active ingredients. Examples ofhot tasting and/or salivation-inducing substances and/or substanceswhich cause a feeling of warmth and/or a tingling feeling on the skin oron the mucous membranes are: capsaicin, dihydrocapsaicin, gingerol,paradol, shogaol, piperine, carboxylic acid-N-vanillylamides, inparticular nonanoic acid-N-vanillylamide, pellitorine or spilanthol,2-nonanoic acid amides, in particular 2-nonanoic acid-N-isobutylamide,2-nonanoic acid-N-4-hydroxy-3-methoxyphenylamide, alkyl ethers of4-hydroxy-3-methoxybenzyl alcohol, in particular4-hydroxy-3-methoxybenzyl-n-butylether, alkyl ethers of4-acyloxy-3-methoxybenzyl alcohol, in particular4-acetyloxy-3-methoxybenzyl-n-butylether and4-acetyloxy-3-methoxybenzyl-n-hexylether, alkyl ethers of3-hydroxy-4-methoxybenzyl alcohol, alkyl ethers of 3,4-dimethoxybenzylalcohol, alkyl ethers of 3-ethoxy-4-hydroxybenzyl alcohol, alkyl ethersof 3,4-methylene dioxybenzyl alcohol, (4-hydroxy-3-methoxyphenyl)aceticacid amides, in particular (4-hydroxy-3-methoxyphenyl)aceticacid-N-n-octylamide, vanillomandelic acid alkylamides, ferulicacid-phenethylamides, nicotinaldehyde, methylnicotinate,propylnicotinate, 2-butoxyethylnicotinate, benzylnicotinate,1-acetoxychavicol, polygodial and isodrimeninol. Hot tasting naturalextracts and/or natural extracts which cause a feeling of warmth and/ora tingling feeling on the skin or on the mucous membranes and which canbe a constituent of a complete flavor composition are: extracts ofpaprika, extracts of pepper (for example capsicum extract), extracts ofchili pepper, extracts of ginger roots, and the like.

As stated hereinabove, any one or a combination of these ingredients maybe added to the slurry during reaction flavor formation, or they may beblended with the reaction flavor solid composition, once the latter isformed in accordance with a method according to the invention.

In addition to the aforementioned ingredients, a complete flavorcomposition may contain carrier materials. Carrier materials areemployed, particularly when the reaction complete flavor composition ispresented in the form of a powder, as flow aids, or extenders, or toprovide physical stability to the powder by modifying the glasstransition temperature (Tg) of the powder. Suitable carriers which maybe included as a component of the reaction flavor solid composition assuch, or as a component in a complete flavor compositions include butare not limited to sugars, sugar derivatives, modified starches,proteins, alcohols, celluloses, dextrins, gums, sugar polyols, peptides,acids, carbohydrates, hydrocolloids. Particular examples of suitablematerials include sugars such as gum arabic, capsul, maltose, sucrose,glucose, lactose, levulose, trehalose, fructose, ribose, dextrose,isomalt, sorbitol, mannitol, xylitol, lactitol, maltitol, pentatol,arabinose, pentose, xylose, galactose; hydrogenated starch hydrolysates,inulin, oligosaccharides such as oligo fructose; maltodextrins ordextrins (i.e., soluble fiber); modified starch; sugar fruit gran; cornsyrup solids; sugar white gran; hydrocolloids such as agar, gum acacia,modified gum acacia, sodium alginate, potassium alginate, ammoniumalginate, calcium alginate or carrageenan; gums; polydextrose;celluloses such as sodium carboxymethylcellulose, enzymaticallyhydrolyzed carboxy methyl cellulose, methyl cellulose, hydroxypropylcellulose and hydroxypropyl methyl cellulose; proteins such as gelatine,pea protein, soy and whey protein isolates and hydrolyzates, and sodiumcasemates; silicon dioxide; and derivatives and mixtures thereof.

Carriers may be employed in complete flavor compositions in amounts of 5to 25 wt % based on the dry weight of the reaction flavor solidcomposition.

Within the scope of this invention are foods or beverages containing areaction flavor solid composition of this invention, alone or as a partof a complete flavor composition.

A feature of the present invention is that the reaction flavorcomposition is a product that is formed externally of a food or beveragematrix. It is an article of manufacture that can impart to, or modify orimprove the flavor of a food or beverage, either alone or as part of acomplete flavor composition, by virtue of it being mixed with or appliedto a food or a beverage. The reaction flavor solid composition is notformed in or on a food or beverage matrix during the cooking process fora food or beverage.

The method may further include a heat-treatment and/or concentratingstep. Such treatments include, without limitation, heating the reactionflavor composition by heating by any method known in the art. Suitableheating means can be selected from conventional means and optionallyinclude a reaction kettle, drum dryer, a flash dryer, a hot plate, anextruder/heater, a concentrator, and other such conventionally knowntechniques. The heat treatment can be carried out at normal pressures orunder increased pressures. For example, a suitable range of heatingtemperature is 80 to 150° C., or 90 to 110° C. Suitable temperatureranges can be from 90 to 150° C., 100 to 150° C., 110 to 150° C., 120 to150° C., 130 to 150° C., or 140 to 150° C.; for can be from 80 to 180°C., 100 to 180° C., 120 to 180° C., 120 to 180° C., 130 to 180° C., 140to 180° C., 150 to 180° C., or 160 to 180° C. At highest temperatureranges, above 150° C. or more, unpleasant burnt tastes may appear.Before, during, or after the heating step, the reaction flavorcomposition may be concentrated (e.g., to dewater the material), bymethods known in the art. In embodiments, the heating and concentrationstep may be carried out concurrently. In embodiments, the remainingmoisture after the heating step may be 30% by weight or less, 25% byweight or less, 20% by weight or less, 15% by weight or less, or 10% byweight or less; or between 20 and 30% by weight, or between 10 and 20%by weight or less. In other embodiments, the volume of the umami tastematerial can be reduced about 50%, about 70%, about 80%, about 90% orabout 95%.

Although the amount of a particular reaction flavor solid compositionemployed in a food or beverage will be dependent upon the intendedapplication and effect that is desired to be achieved, generally, anamount of 0.1 to 1% by weight and preferably, about 0.1 to 0.5% byweight is appropriate to impart a desirable flavor and/or aroma to afood or beverage, or modify or improve the flavor and/or aroma of a foodor beverage.

Examples of foods or beverages include baked products, snack foods,cereal products, alcoholic and non-alcoholic beverages, spice blends,ready-to-heat foods, ready-to-eat meals, dairy products, meat products,seasoning preparations, ketchup, sauces, dried vegetables, soups,bouillon, noodles, frozen entrees, gravy, and desserts. Reaction flavorsolid compositions of the present invention can make a generalimprovement to the flavor of foods or beverages. The reaction flavorsolid compositions may be added to a food or beverage by simple mixingwith other ingredients in the final blending of a food or beverage, suchas a convenience food.

Alternatively, the reaction flavor solid composition may be added to theoutside of a food or beverage, for example, the process of dusting orspray coating a snack food. Still further, the reaction flavor solidcomposition may be added to a food or beverage during its formation, ina process which is sometimes referred to as internal flavoring.

The reaction flavor solid compositions of the present invention arewell-suited for use, without limitation, in any edible product, such asthose products disclosed herein.

In order to make the food compositions of the present invention, themethod includes a step of providing a myceliated high-protein foodproduct. Additionally, the food compositions of the present invention,in an embodiment, comprise a myceliated high-protein food product. Thepresent inventors have previously disclosed a method to prepare amyceliated high-protein food product, which includes culturing afilamentous fungus in an aqueous media which has a high level ofprotein, for example at least 20% protein (w/w), on a dry weight basis,and the media contains at least 50 g/L protein. The fungi can includeLentinula edodes, Agaricus spp., Pleurotus spp., Boletus spp., orLaetiporus spp.; e.g., Pleurotus ostreatus, Pleurotus eryngii, Lepistanuda, Hericium erinaceus, Lentinula edodes, Agaricus blazeii, Cordycepssinensis, Laetiporus sulfureus and combinations thereof. Compositionscomprising a myceliated high-protein food product are also disclosed.See, e.g., U.S. Pat. No. 10,010,103, filed Apr. 14, 2017, U.S. Ser. No.16/025,365, (filed Jul. 2, 2018), both entitled “Methods for theProduction and use of Myceliated High-Protein Food Compositions,”, U.S.Ser. No. 62/752,158 (filed Oct. 29, 2018), U.S. Ser. No. 62/796,438(filed Jan. 24, 2019), related to aqueous-phase fermentation of proteinmaterials, all of which are incorporated by reference herein in theirentireties.

In one embodiment, the present invention includes a method step toprepare a myceliated high-protein food product. The method mayoptionally include the steps of providing an aqueous media comprising ahigh-protein material. The aqueous media may comprise, consist of, orconsist essentially of at least 20% protein (w/w), on a dry weightbasis, and is 50 g/L protein. The media may also comprise, consist of orconsist essentially of optional additional excipients as identifiedherein below. The aqueous media may be inoculated with a fungal culture.The inoculated media may then be cultured to produce a myceliatedhigh-protein food product, and the myceliated high-protein food producttaste, flavor, and/or aroma may be modulated compared to thehigh-protein material in the absence of the culturing step.

The aqueous media may comprise, consist of, or consist essentially of ahigh-protein material. The high-protein material to include in theaqueous media can be obtained from a number of sources, includingvegetarian sources (e.g., plant sources) as well as non-vegetariansources, and can include a protein concentrate and/or isolate.Vegetarian sources include meal, protein concentrates and isolatesprepared from a vegetarian source such as pea, rice, soy, cyanobacteria,grain, hemp, chia, chickpea, potato protein, algal protein and nettleprotein or combinations of these. In embodiments, the vegetarian sourceis pea, rice, chickpea or a combination thereof. In embodiments, thevegetarian source is pea, chickpea or a combination thereof. Inembodiments, the vegetarian source is rice, chickpea, or a combinationthereof. Typically, a protein concentrate is made by removing the oiland most of the soluble sugars from a meal, such as soybean meal. Such aprotein concentrate may still contain a significant portion ofnon-protein material, such as fiber. Typically, protein concentrationsin such products are between 55-90%. The process for production of aprotein isolate typically removes most of the non-protein material suchas fiber and may contain up to about 90-99% protein. A typical proteinisolate is typically subsequently dried and is available in a powderedform and may alternatively be called “protein powder.”

Non-vegetarian sources for the high-protein material may also be used inthe present invention. Such non-vegetarian sources include whey, casein,egg, meat (beef, chicken, pork sources, for example), isolates,concentrates, broths, or powders.

In one embodiment, mixtures of any of the high-protein materialsdisclosed herein can be used to provide, for example, favorablequalities, such as a more complete (in terms of amino acid composition)high-protein material. In one embodiment, high-protein materials such aspea protein and rice protein can be combined. In one embodiment, theratio of a mixture can be from 1:10 to 10:1 pea protein:rice protein (ona dry basis). In one embodiment, the ratios can optionally be 5:1 to1:5, 2:1 to 1:2, or in one embodiment, 1:1. In other embodiments, theratio can be 65:35.

The high-protein material itself can be about 20% protein, 30% protein,40% protein, 45% protein, 50% protein, 55% protein, 60% protein, 65%protein, 70% protein, 75% protein, 80% protein, 85% protein, 90%protein, 95% protein, or 98% protein, or at least about 20% protein, atleast about 30% protein, at least about 40% protein, at least about 45%protein, at least about 50% protein, at least about 55% protein, atleast about 60% protein, at least about 65% protein, at least about 70%protein, at least about 75% protein, at least about 80% protein, atleast about 85% protein, at least about 90% protein, at least about 95%protein, or at least about 98% protein.

This invention discloses the use of concentrated media, which provides,for example, an economically viable economic process for production ofan acceptably tasting and/or flavored high-protein food product. In oneembodiment of the invention the total media concentration is up to 150g/L but can also be performed at lower levels, such as 5 g/L. Higherconcentrations in media result in a thicker and/or more viscous media,and therefore are optionally processed by methods known in the art toavoid engineering issues during culturing or fermentation. To maximizeeconomic benefits, a greater amount of high-protein material per L mediais used. The amount is used is chosen to maximize the amount ofhigh-protein material that is cultured, while minimizing technicaldifficulties in processing that may arise during culturing such asviscosity, foaming and the like.

The amount of total protein in the aqueous media may comprise, consistof, or consist essentially of at least 20 g, 25 g, 30 g, 35 g, 40 g, 45g, 50 g, 55 g, 60 g, 65 g, 70 g, 75 g, 80 g, 85 g, 90 g, 95 g, or 100 g,or more, of protein per 100 g total dry weight w/w, or per total allcomponents on a dry weight basis. Alternatively, the amount of proteincomprises, consist of, or consist essentially of between 20 g to 90 g,between 30 g and 80 g, between 40 g and 70 g, between 50 g and 60 g, ofprotein per 100 g dry weight w/win the media.

In some embodiments, the total protein in aqueous media is about 45 g toabout 100 g, or about 80-100 g of protein per 100 g dry weight w/w.

In another embodiment, the aqueous media comprises between about 1 g/Land 200 g/L, between about 5 g/L and 180 g/L, between about 20 g/L and150 g/L, between about 25 g/L and about 140 g/L, between about 30 g/Land about 130 g/L, between about 35 g/L and about 120 g/L, between about40 g/L and about 110 g/L, between about 45 g/L and about 105 g/L,between about 50 g/L and about 100 g/L, between about 55 g/L and about90 g/L, or about 75 g/L protein; or between about 50 g/L-150 g/L, orabout 75 g/L and about 120 g/L, or about 85 g/L and about 100 g/L.Alternatively, the aqueous media comprises at least about 10 g/L, atleast about 15 g/L, at least about 20 g/L, at least about 25 g/L, atleast about 30 g/L, at least about 35 g/L, at least about 40 g/L or atleast about 45 g/L protein. In fermenters, in some embodiments theamount to use includes between about 1 g/L and 150 g/L, between about 10g/L and 140 g/L, between about 20 g/L and 130 g/L, between about 30 g/Land about 120 g/L, between about 40 g/L and about 110 g/L, between about50 g/L and about 100 g/L, between about 60 g/L and about 90 g/L, betweenabout 70 g/L and about 80 g/L, or at least about 20 g/L, at least about30 g/L, at least about 40 g/L, at least about 50 g/L, at least about 60g/L, at least about 70 g/L, at least about 80 g/L, at least about 90g/L, at least about 100 g/L, at least about 110 g/L, at least about 120g/L, at least about 130 g/L or at least about 140 g/L.

In some embodiments, the aqueous media comprises between about 50 g/Land about 100 g/L, or about 80 g/L, about 85 g/L, about 90 g/L, about 95g/L about 100 g/L, about 110 g/L, about 120 g/L, about 130 g/L, about140 g/L, or about 150 g/L.

In some embodiments, the high-protein material, after preparing theaqueous media of the invention, is not completely dissolved in theaqueous media. Instead, the high-protein material may be partiallydissolved, and/or partially suspended, and/or partially colloidal.However, even in the absence of complete dissolution of the high-proteinmaterial, positive changes may be affected during culturing of thehigh-protein material. In one embodiment, the high-protein material inthe aqueous media is kept as homogenous as possible during culturing,such as by ensuring agitation and/or shaking.

In one embodiment, the aqueous media further comprises, consists of, orconsists essentially of materials other than the high-protein material,e.g., excipients as defined herein and/or in particular embodiments.Excipients can comprise any other components known in the art topotentiate and/or support fungal growth, and can include, for example,nutrients, such as proteins/peptides, amino acids as known in the artand extracts, such as malt extracts, meat broths, peptones, yeastextracts and the like; energy sources known in the art, such ascarbohydrates; essential metals and minerals as known in the art, whichincludes, for example, calcium, magnesium, iron, trace metals,phosphates, sulphates; buffering agents as known in the art, such asphosphates, acetates, and optionally pH indicators (phenol red, forexample). Excipients may include carbohydrates and/or sources ofcarbohydrates added to media at 5-10 g/L. It is usual to add pHindicators to such formulations.

Excipients may also include peptones/proteins/peptides, as is known inthe art. These are usually added as a mixture of protein hydrolysate(peptone) and meat infusion, however, as used in the art, theseingredients are typically included at levels that result in much lowerlevels of protein in the media than is disclosed herein. Many mediahave, for example, between 1% and 5% peptone content, and between 0.1and 5% yeast extract and the like.

In one embodiment, excipients include for example, yeast extract, maltextract, maltodextrin, peptones, and salts such as diammonium phosphateand magnesium sulfate, as well as other defined and undefined componentssuch as potato or carrot powder. In some embodiments, organic (asdetermined according to the specification put forth by the NationalOrganic Program as penned by the USDA) forms of these components may beused.

In one embodiment, excipients comprise, consist of, or consistessentially of dry carrot powder, dry malt extract, diammoniumphosphate, magnesium sulfate, and citric acid. In one embodiment,excipients comprise, consist of, or consist essentially of dry carrotpowder between 0.1-10 g/L, dry malt extract between 0.1 and 20 g/L,diammonium phosphate between 0.1 and 10 g/L, and magnesium sulfatebetween 0.1 and 10 g/L. Excipients may also optionally comprise, consistof, or consist essentially of citric acid and an anti-foam component.The anti-foam component can any anti-foam component known in the art,such as a food-grade silicone anti-foam emulsion or an organic polymeranti-foam (such as a polypropylene-based polyether composition).

In another embodiment, the medium comprises, consists of or consistsessentially of the high protein material as defined herein and ananti-foam component, without any other excipients present.

The method may also comprise the optional step of sterilizing theaqueous media prior to inoculation by methods known in the art,including steam sterilization and all other known methods to allow forsterile procedure to be followed throughout the inoculation andculturing steps to enable culturing and myceliation by pure fungalstrains. Alternatively, the components of the media may be separatelysterilized and the media may be prepared according to sterile procedure.

The method also includes inoculating the media with a fungal culture.The fungal culture may be prepared by culturing by any methods known inthe art. In one embodiment, the methods to culture may be found in,e.g., PCT/US14/29989, filed Mar. 15, 2014, PCT/US14/29998, filed Mar.15, 2014, all of which are incorporated by reference herein in theirentireties.

The fungal cultures, prior to the inoculation step, may be propagatedand maintained as is known in the art. In one embodiment, the fungidiscussed herein can be kept on 2-3% (v/v) fruit puree with 3-4% agar(m/v). Such media is typically prepared in 21.6 L handled glass jarsbeing filled with 1.4-1.5 L media. Such a container pours for 50-60 90mm Petri plates. The media is first sterilized by methods known in theart, typically with an autoclave. Conventional B. stearothermophilus andthermocouple methods are used to verify sterilization parameters. Somestrains, such as L. sulfureus, grow better when supplemented with 1%yellow cornmeal. Agar media can also be composed of high-proteinmaterial to sensitize the strain to the final culture. This techniquemay also be involved in strain selection of the organisms discussedherein. Agar media should be poured when it has cooled to the pointwhere it can be touched by hand (˜40-50° C.).

In one embodiment, maintaining and propagating fungi for use forinoculating the high-protein material as disclosed in the presentinvention may be carried out as follows. For example, a propagationscheme that can be used to continuously produce material according tothe methods is discussed herein. Once inoculated with master culture andsubsequently colonized, Petri plate cultures can be used at any point topropagate mycelium into prepared liquid media. As such, plates can bepropagated at any point during log phase or stationary phase but areencouraged to be used within three months and in another embodimentwithin 2 years, though if properly handled by those skilled in the artcan generally be stored for as long as 10 years at 4° C. and up to 6years at room temperature.

In some embodiments, liquid cultures used to maintain and propagatefungi for use for inoculating the high-protein material as disclosed inthe present invention include undefined agricultural media with optionalsupplements as a motif to prepare culture for the purposes ofinoculating solid-state material or larger volumes of liquid. As such,liquid media are typically inoculated with agar, liquid and other formsof culture. Bioreactors provide the ability to monitor and controlaeration, foam, temperature, and pH and other parameters of the cultureand as such enables shorter myceliation times and the opportunity tomake more concentrated media.

In one embodiment, the fungi for use for inoculating the high-proteinmaterial as disclosed in the present invention may be prepared as asubmerged liquid culture and agitated on a shaker table, or may beprepared in a shaker flask, by methods known in the art and according tomedia recipes disclosed in the present invention. In one embodiment, thefungal component may be prepared from a glycerol stock, by a simplepropagation motif of Petri plate culture to 0.5-4 L Erlenmeyer shakeflask to 50% glycerol stock. Petri plates can comprise agar in 10-35 g/Lin addition to various media components. Conducted in sterile operation,chosen Petri plates growing anywhere from 1-˜3,652 days can bepropagated into 0.5-4 L Erlenmeyer flasks (or 250 to 1,000 mL Wheatonjars, or any suitable glassware) for incubation on a shaker table orstationary incubation. The smaller the container, the faster the shakershould be. In one embodiment, the shaking is anywhere from 40-160 RPMdepending on container size and, with about a 1″ swing radius.

Liquid-state fermentation agitation and swirling techniques as known inthe art are also employed which include mechanical shearing usingmagnetic stir bars, stainless steel impellers, injection of sterilehigh-pressure air, the use of shaker tables and other methods such aslighting regimen, batch feeding or chemostatic culturing, as known inthe art.

In one embodiment, culturing step is carried out in a bioreactor whichis ideally constructed with a torispherical dome, cylindrical body, andspherical cap base, jacketed about the body, equipped with a magneticdrive mixer, and ports to provide access for equipment comprising DO,pH, temperature, level and conductivity meters as is known in the art.Any vessel capable of executing the methods of the present invention maybe used. In another embodiment the set-up provides 0.1-5.0 ACH. Otherengineering schemes known to those skilled in the art may also be used.

The reactor can be outfitted to be filled with sterile water. In oneembodiment the entire media is sterilized in situ while in anotherembodiment concentrated media is sterilized and diluted into a vesselfilled water that was filter and/or heat sterilized, or sufficientlytreated so that it doesn't encourage contamination over the colonizingfungus. In another embodiment, high temperature high pressuresterilizations are fast enough to be not detrimental to the media. Inone embodiment the entire media is sterilized in continuous mode byapplying high temperature between 130° and 150° C. for a residence timeof 1 to 15 minutes. Once prepared with a working volume of sterilemedia, the tank can be mildly agitated and inoculated. Either as aconcentrate or whole media volume in situ, the media can be heatsterilized by steaming either the jacket, chamber or both while themedia is optionally agitated. The medium may optionally be pasteurizedinstead.

In one embodiment, the reactor is used at a large volume, such as in500,000-200,000 L working volume bioreactors. When preparing material atsuch volumes the culture must pass through a successive series of largerbioreactors, any bioreactor being inoculated at 0.5-15% of the workingvolume according to the parameters of the seed train. A typical processwould pass a culture from master culture, to Petri plates, to flasks, toseed bioreactors to the final main bioreactor when scaling the method ofthe present invention. To reach large volumes, 3-4 seeds may be used.The media of the seed can be the same or different as the media in themain. In one embodiment, the fungal culture for the seed is a proteinconcentration as defined herein, to assist the fungal culture inadapting to high-protein media in preparation for the main fermentation.Such techniques are discussed somewhat in the examples below. In oneembodiment, foaming is minimized by use of anti-foam on the order of 0.5to 2.5 g/L of media, such as those known in the art, including insolubleoils, polydimethylsiloxanes and other silicones, certain alcohols,stearates and glycols. In one embodiment, lowering pH assists in culturegrowth, for example, for L. edodes pH may be adjusted by use of citricacid or by any other compound known in the art, but care must be takento avoid a sour taste for the myceliated high-protein product. The pHmay be adjusted to between about 4.5 and 5.5, for example, to assist ingrowth.

In one embodiment, during the myceliation step, for example, wherein themedia comprises at least 50% (w/w) protein on a dry weight basis, and/orwherein the media comprises at least 50 g/L protein, the pH does notchange during processing. “pH does not change during processing” isunderstood to mean that the pH does not change in any significant way,taking into account variations in measured pH which are due toinstrument variations and/or error. For example, the pH will stay withinabout plus or minus 0.3 pH units, plus or minus 0.25 pH units, plus orminus 0.2 pH units, plus or minus 0.15 pH units, or plus or minus 0.1 pHunits of a starting pH of the culture during the myceliation, e.g.processing step. Minor changes in pH are also contemplated duringprocessing, particularly in media which do not contain an exogenousbuffer such as diammonium phosphate. A minor change in pH can be definedas a pH change of plus or minus 0.5 pH units or less, plus or minus 0.4pH units or less, plus or minus 0.3 pH units or less, plus or minus 0.25pH units or less, plus or minus 0.2 pH units or less, plus or minus 0.15pH units or less, or plus or minus 0.1 pH units or less of a startingpH.

In an exemplification of the preparation of L. edodes as the fungalcomponent for use for inoculating an aqueous media to prepare themyceliated high-protein food product, 1:1 mixture of pea protein andrice protein at 40% protein (8 g per 20 g total plus excipients) mediawas prepared, and the increase in biomass concentration was correlatedwith a drop in pH. After shaking for 1 to 10 days, an aliquot (e.g. 10to 500 mL) of the shake flask may be transferred in using sterileprocedure into a sterile, prepared sealed container (such as acustomized stainless steel can or appropriate conical tube), which canthen adjusted with about 5-60%, sterile, room temperature (v/v)glycerol. The glycerol stocks can may be sealed with a water tight sealand can be held stored at −20° C. for storage. The freezer is ideally aconstant temperature freezer. Glycerol stocks stored at 4° C. may alsobe used. Agar cultures can be used as inoculant for the methods of thepresent invention, as can any culture propagation technique known in theart.

It was found that not all fungi are capable of growing in media asdescribed herein. Fungi useful for the present invention are from thehigher order Basidio- and Ascomycetes. In some embodiments, fungieffective for use in the present invention include, but are not limitedto, Lentinula spp., such as L. edodes, Agaricus spp., such as A. blazei,A. bisporus, A. campestris, A. subrufescens, A. brasiliensis, or A.silvaticus; Pleurotus spp., Boletus spp., or Laetiporus spp. In oneembodiment, the fungi for the invention include fungi from optionally,liquid culture of species generally known as oyster, porcini, ‘chickenof the woods’ and shiitake mushrooms. These include Pleurotus (oyster)species such as Pleurotus ostreatus, Pleurotus salmoneostramineus(Pleurotus djamor), Pleurotus eryngii, or Pleurotus citrinopileatus;Boletus (porcini) species such as Boletus edulis; Laetiporus (chicken ofthe woods) species such as Laetiporus sulfureus, and many others such asL. budonii, L. miniatus, L. flos-musae, L. discolor; and Lentinula(shiitake) species such as L. edodes. Also included are Lepista nuda,Hericium erinaceus, Agaricus blazeii, and combinations thereof. In oneembodiment, the fungi is Lentinula edodes.

In other embodiments, the filamentous fungus may comprise, consist of,or consist essentially of Hericium erinaceus, Lentinula edodes,Cantharellus cibarius, Cordyceps sinensis, Ganodenna lucidum, Laetiporussulphureus, Laetiporus cincinnatus, Morchella angusticeps, Morchellaimportuna, Grifola frondosa, Grifola curtisii, Pleurotus ostreatus,Pleurotus umbellatus, Volvariella volvacea, Pleurotus salmoneostramineus(Pleurotus djamor), Pleurotus eryngii, Pleurotus citrinopileatus,Cantherellus cibarius (chanterelle), Fumaria officianalis, Fistulinahepatica, Sparassis crispa, Inonotus obliquus, Cordyceps militaris,Cyclocybe aegerita, Flammulina velutipes, Morchella esculenta,Laetiporus cincinnatus, Morchella importuna, Hypsizygus tessellatus,Stropharia Rugoso-Annulata, Termitomyces albuminosus, and/orcombinations thereof.

Fungi may be obtained commercially, for example, from the Penn StateMushroom Culture Collection. Strains are typically received as “masterculture” PDY slants in 50 mL test tubes and are stored at all, but forA. blazeii, stored at 4° C. until plated. For plating, small pieces ofculture are typically transferred into sterile shake flasks (e.g. 250mL) so as not to contaminate the flask filled with a sterilized media(liquid media recipes are discussed below). Inoculated flasks shake forapproximately ten hours and aliquots of said flasks are then plated ontoprepared Petri plates of a sterile agar media. One flask can be used toprepare dozens to potentially hundreds of Petri plate cultures. Thereare other methods of propagating master culture though the inventorsfind these methods as disclosed to be simple and efficient.

Determining when to end the culturing step and to harvest the myceliatedhigh-protein food product, which according to the present invention, toresult in a myceliated high-protein food product with acceptable taste,flavor and/or aroma profiles, can be determined in accordance with anyone of a number of factors as defined herein, such as, for example,visual inspection of mycelia, microscope inspection of mycelia, pHchanges, changes in dissolved oxygen content, changes in proteincontent, amount of biomass produced, and/or assessment of taste profile,flavor profile, or aroma profile. In one embodiment, harvest can bedetermined by tracking protein content during culturing and harvestbefore significant catabolism of protein occurs. The present inventorsfound that protein catabolism can initiate in bioreactors at 30-50 hoursof culturing under conditions defined herein. In another embodiment,production of a certain amount of biomass may be the criteria used forharvest. For example, biomass may be measured by filtering, such througha filter of 10-1000 μm, and has a protein concentration between 0.1 and25 g/L; or in one embodiment, about 0.2-0.4 g/L. In one embodiment,harvest can occur when the dissolved oxygen reaches about 10% to about90% dissolved oxygen, or less than about 80% of the starting dissolvedoxygen. Additionally, mycelial products may be measured as a proxy formycelial growth, such as, total reducing sugars (usually a 40-95%reduction), β-glucan and/or chitin formation; harvest is indicated at102-104 ppm. Other indicators include small molecule metaboliteproduction depending on the strain (e.g. eritadenine on the order of0.1-20 ppm for L. edodes or erinacine on the order of 0.1-1,000 ppm forH. erinaceus) or nitrogen utilization (monitoring through the use of anynitrogenous salts or protein, cultures may be stopped just as proteinstarts to get utilized or may continue to culture to enhance thepresence of mycelial metabolites). In one embodiment, the total proteinyield in the myceliated high-protein food product after the culturingstep is about 75% to about 95%.

Harvest includes obtaining the myceliated high-protein food productwhich is the result of the myceliation step. After harvest, cultures canbe processed according to a variety of methods. In one embodiment, themyceliated high-protein food product is pasteurized or sterilized. Inone embodiment, the myceliated high-protein food product is driedaccording to methods as known in the art. Additionally, concentrates andisolates of the material may be prepared using variety of solvents orother processing techniques known in the art. In one embodiment thematerial is pasteurized or sterilized, dried and powdered by methodsknown in the art. Drying can be done in a desiccator, vacuum dryer,conical dryer, spray dryer, fluid bed or any method known in the art.Preferably, methods are chosen that yield a dried myeliated high-proteinproduct (e.g., a powder) with the greatest digestibility andbioavailability. The dried myeliated high-protein product can beoptionally blended, pestled milled or pulverized, or other methods asknown in the art.

In many cases, the flavor, taste and/or aroma of high-protein materialsas disclosed herein, such as protein concentrates or isolates fromvegetarian or nonvegetarian sources (e.g. egg, whey, casein, beef, soy,rice, hemp, pea, chickpea, soy, cyanobacteria, and chia) may haveflavors, which are often perceived as unpleasant, having pungent aromasand bitter or astringent tastes. These undesirable flavors and tastesare associated with their source(s) and/or their processing, and theseflavors or tastes can be difficult or impossible to mask or disguisewith other flavoring agents. The present invention, as explained in moredetail below, works to modulate these tastes and/or flavors.

In one embodiment of the invention, flavors and/or tastes of themyceliated high-protein food product or products are modulated ascompared to the high-protein material (starting material). In someembodiments, both the sterilization and myceliation contribute to themodulation of the resultant myceliated high-protein food products'taste.

In one embodiment, the aromas of the resultant myceliated high-proteinfood products prepared according to the invention are reduced and/orimproved as compared to the high-protein material (starting material).In other words, undesired aromas are reduced and/or desired aromas areincreased. In another embodiment, flavors and/or tastes may be reducedand/or improved. For example, desirable flavors and/or tastes may beincreased or added to the high-protein material by the processes of theinvention, resulting in myceliated high-protein food products that haveadded mushroom, meaty, umami, popcorn, buttery, and/or other flavors ortastes to the food product. The increase in desirable flavors and/ortastes may be rated as an increase of 1 or more out of a scale of 5 (1being no taste, 5 being a very strong taste.)

Flavors and/or tastes of myceliated high-protein food products may alsobe improved by processes of the current invention. For example,deflavoring can be achieved, resulting in a milder flavor and/or withthe reduction of, for example, bitter and/or astringent tastes and/orbeany and/or weedy and/or grassy tastes. The decrease in undesirableflavors and/or tastes as disclosed herein may be rated as a decrease of1 or more out of a scale of 5 (1 being no taste, 5 being a very strongtaste.)

Culturing times and/or conditions can be adjusted to achieve the desiredaroma, flavor and/or taste outcomes. For example, cultures grown forapproximately 2-3 days can yield a deflavored product whereas culturesgrown for longer may develop various aromas that can change/intensify asthe culture grows. As compared to the control and/or high-proteinmaterial, and/or the pasteurized, dried and powdered medium notsubjected to sterilization or myceliation, the resulting myceliatedhigh-protein food product in some embodiments is less bitter and has amilder, less beany aroma.

In one embodiment of the present invention, the myceliated high-proteinfood products made by the methods of the invention have a complete aminoacid profile (all amino acids in the required daily amount) because ofthe media from which it was made has such a profile. While amino acidand amino acid profile transformations are possible according to themethods of the present invention, many of the products made according tothe methods of the present invention conserve the amino acid profilewhile at the same time, more often altering the molecular weightdistribution of the proteome.

In one embodiment, when grown in a rice and pea protein concentratemedium the oyster fungi (Pleurotus ostreatus) can convey a strong savoryaroma that leaves after a few seconds at which point a mushroom flavoris noticeable. In one embodiment, the strains convey a savory meatyaroma and/or umami, savory or meaty flavor and/or taste. L. edodes andA. blazeii in some embodiments are effective at deflavoring with shorterculturing times, such as 1.5-8 days, depending on whether the culture isin a shake flask or bioreactor. L. edodes is particularly good for thedeflavoring of pea and rice protein concentrate mixtures.

The present invention discloses production of a food compositioncomprising the myceliated food product made by any of the methods of asdisclosed herein, which is then used to mix with other edible componentsto provide the food compositions as disclosed herein. Alternatively, theinvention comprises a food composition for human consumption, comprisinga myceliated high-protein food product, myceliated high-protein foodproduct, wherein the myceliated high-protein food product is at least50% (w/w) protein on a dry weight basis, wherein the myceliatedhigh-protein product is myceliated by an aqueous fungal culture, in amedia comprising at least 50 g/L protein in liquid culture; and anedible material. A myceliated high protein food product of the presentinvention is also referred to herein as PURETASTE™ protein, PT, PTP, andthe like.

The present invention also comprises a food composition comprising amixture of a myceliated high-protein food product as defined herein andan edible material. The food composition can comprise, consist of, orconsist essentially of at least 10%, at least 20%, at least 25%, atleast 30%, at least 35%, at least 40%, at least 45%, at least 50%, atleast 55%, at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, or at least 95%, protein.

“Myceliated” as used herein, means a high-protein material as definedherein having been cultured with live fungi as defined herein andachieved at least a 1%, at least 2%, at least 3%, at least 4%, at leasta 5%, at least a 10%, at least a 20%, at least a 30%, at least a 40%, atleast a 50%, at least a 60%, at least a 70%, at least a 80%, at least a90%, at least a 100%, at least a 120%, at least a 140%, at least a 160%,at least a 180%, at least a 200%, at least a 250%, at least a 300%, atleast a 400%, at least a 500% increase in biomass or more, to result ina myceliated high-protein food product. Alternatively, “myceliated” mayrefer to the distribution of a previously-grown biomass from afilamentous fungus as disclosed herein through the high-proteinmaterial.

In some embodiments, the high-protein material is a protein concentrateor a protein isolate, which may be obtained from vegetarian ornonvegetarian source as defined herein, including pea, rice, chickpea,or combinations thereof. In some embodiments, the myceliatedhigh-protein food product can be myceliated by a fungal culture asdefined herein. In some embodiments, the myceliated high-protein foodproduct can have enhanced meaty, savory, umami, popcorn, and/or mushroomflavors, aromas and/or tastes as compared to the high-protein material.In other embodiments, the myceliated high-protein food product hasdecreased flavors, tastes and/or aromas (deflavoring) leading to amilder and/or an improved flavor, taste or aroma. In one embodimentreduced bitterness, astringency and/or beany, grassy or weedy tastes areobserved.

The following examples further illustrate the invention but, of course,should not be construed as in any way limiting its scope.

Example 1

Eighteen (18) 1 L baffled DeLong Erlenmeyer flasks were filled with0.400 L of a medium consisting of 25 g/L organic pea protein concentrate(labeled as 80% protein), 25 g/L organic rice protein concentrate(labeled as 80% protein), 4 g/L organic dry malt extract, 2 g/Ldiammonium phosphate, 1 g/L organic carrot powder and 0.4 g/L magnesiumsulfate heptahydrate in RO water. The flasks were covered with astainless steel cap and sterilized in an autoclave on a liquid cyclethat held the flasks at 120-121° C. for 90 minutes. The flasks werecarefully transferred to a clean HEPA laminar flowhood where they cooledfor 18 hours. Sixteen (16) flasks were subsequently inoculated with 2cm² pieces of mature Petri plate cultures of P. ostreatus, P. eryngii,L. nuda, H. erinaceus, L. edodes, A. blazeii, L. sulfureus and B.edulis, each strain done in duplicate from the same plate. All 18 flaskswere placed on a shaker table at 150 rpm with a swing radius of 1″ atroom temperature. All samples showed reduced pea and reduced rice aromaand flavor, as well as less “beany” type aromas/flavors.

Example 2

Three (3) 4 L Erlenmeyer flasks were filled with 1.5 L of a mediumconsisting of 5 g/L pea protein concentrate (labeled as 80% protein), 5g/L rice protein concentrate (labeled as 80% protein), 3 g/L maltextract and 1 g/L carrot powder. The flasks were wrapped with asterilizable biowrap which was wrapped with autoclave tape 5-6 times(the taped biowrap should be easily taken off and put back on the flaskwithout losing shape) and sterilized in an autoclave that held theflasks at 120-121° C. for 90 minutes. The flasks were carefullytransferred to a clean HEPA laminar flowhood where they cooled for 18hours. Each flask was subsequently inoculated with pieces of P1 Petriplate cultures of L. edodes and placed on a shaker table at 120 rpm at26° C. After 7-15 days, the inventors noticed, by using a pH probe on 20mL culture aliquots, that the pH of every culture had dropped nearly 2points since inoculation. L. edodes is known to produce various organicacids on or close to the order of g/L and the expression of these acidsare likely what dropped the pH in these cultures. A microscope check wasdone to ensure the presence of mycelium and the culture was plated on LBmedia to ascertain the extent of any bacterial contamination. While thisculture could have been used as a food product with further processing(pasteurization and optionally drying), the inventors typically use suchcultures as inoculant for bioreactor cultures of media prepared asdisclosed according to the methods of the present invention.

Example 3

A 7 L bioreactor was filled with 4.5 L of a medium consisting of 5 g/Lpea protein concentrate (labeled as 80% protein), 5 g/L rice proteinconcentrate (labeled as 80% protein), 3 g/L malt extract and 1 g/Lcarrot powder. Any open port on the bioreactor was wrapped with tinfoiland sterilized in an autoclave that held the bioreactor at 120-121° C.for 2 hours. The bioreactor was carefully transferred to a clean benchin a cleanroom, setup and cooled for 18 hours. The bioreactor wasinoculated with 200-500 mL of inoculant as prepared in Example 2. Thebioreactor was held at 26° C. A kick-in/kick-out anti-foam system wassetup and it was estimated that ˜1.5 g/L anti-foam was added during theprocess. At ˜3-4 days the inventors observed the flask culture. Amicroscope check was done to ensure the presence of mycelium (mycelialpellets were visible by the naked eye) and the culture was plated on LBmedia to ascertain the extent of any bacterial contamination and nonewas observed. While this culture could have been used as a food productwith further processing (pasteurization and optionally drying), theinventors typically use such cultures as inoculant for bioreactorcultures of media prepared as disclosed according to the methods of thepresent invention.

Example 4

A 250 L bioreactor was filled with 150 L of a medium consisting of 45g/L pea protein concentrate (labeled as 80% protein), 45 g/L riceprotein concentrate (labeled as 80% protein), 1 g/L carrot powder, 1.8g/L diammonium phosphate, 0.7 g/L magnesium sulfate heptahydrate, 1 g/Lanti-foam and 1.5 g/L citric acid and sterilized in place by methodsknown in the art, being held at 120-121° C. for 100 minutes. Thebioreactor was inoculated with 5 L of inoculant from two bioreactors asprepared in Example 3. The bioreactor was held at 26° C. The culture washarvested in 4 days upon successful visible (mycelial pellets) andmicroscope checks. The culture was plated on LB media to ascertain theextent of any bacterial contamination and none was observed. The culturewas then pasteurized at 82° C. for 30 minutes with a ramp up time of 30minutes and a cool down time of 45 minutes to 17° C. The culture wasfinally spray dried and tasted. The final product was noted to have amild aroma with no perceptible taste at concentrations up to 10%. Theproduct was ˜75% protein on a dry weight basis.

Example 5

A 250 L bioreactor was filled with 150 L of a medium consisting of 45g/L pea protein concentrate (labeled as 80% protein), 45 g/L riceprotein concentrate (labeled as 80% protein), 1 g/L carrot powder, 1.8g/L diammonium phosphate, 0.7 g/L magnesium sulfate heptahydrate, 1 g/Lanti-foam and 1.5 g/L citric acid and sterilized in place by methodsknown in the art, being held at 120-121° C. for 100 minutes. Thebioreactor was inoculated with 5 L of inoculant from two bioreactors asprepared in Example 3. The bioreactor was held at 26° C. The culture washarvested in 2 days upon successful visible (mycelial pellets) andmicroscope checks. The culture was plated on LB media to ascertain theextent of any bacterial contamination and none was observed. The culturewas then pasteurized at 82° C. for 30 minutes with a ramp up time of 30minutes and a cool down time of 90 minutes to 10° C. The culture wasfinally concentrated to 20% solids, spray dried and tasted. The finalproduct was noted to have a mild aroma with no perceptible taste atconcentrations up to 10%. The product was ˜75% protein on a dry weightbasis.

The amount of lactic acid in the final product (Product Batch 1 and 2are from to different fermentation runs) were as follows, as shown inTable 1:

TABLE 1 Product Lactic Acid Batch (g/L) 1 0.13 2 0.14

Example 6

Eight (8) 1 L baffled DeLong Erlenmeyer flasks were filled with 0.4 L ofmedia consisting of 45 g/L pea protein concentrate (labeled as 80%protein), 45 g/L rice protein concentrate (labeled as 80% protein), 1g/L carrot powder, 1 g/L malt extract, 1.8 g/L diammonium phosphate and0.7 g/L magnesium sulfate heptahydrate and sterilized in an autoclavebeing held at 120-121° C. for 90 minutes. The flasks were then carefullyplaced into a laminar flowhood and cooled for 18 hours. Each flask wasinoculated with 240 mL of culture as prepared Example 2 except thestrains used were G. lucidum, C. sinensis, I. obliquus and H. erinaceus,with two flasks per species. The flasks were shaken at 26° C. at 120 RPMfor 8-15 days, at which point they were pasteurized as according to theparameters discussed in Example 5, desiccated, pestled and tasted. TheG. lucidum product contained a typical ‘reishi’ aroma, which most of thetasters found pleasant. The other samples were deemed pleasant as wellbut had more typical mushroom aromas.

As compared to the control, the pasteurized, dried and powdered mediumnot subjected to sterilization or myceliation, the resulting myceliatedfood products was thought to be much less bitter and to have had a moremild, less beany aroma that was more cereal in character than beany by 5tasters. The sterilized but not myceliated product was thought to haveless bitterness than the non-sterilized control but still had a strongbeany aroma. The preference was for the myceliated food product.

Example 7

Fermentation Operation in 4,000 L Bioreactor Using Continuous Sterilizer

A 4,000 L bioreactor was filled with 2,500 L of a sterilized mediumsimilar to Example 4, consisting of 45 g/L pea protein concentrate(labeled as 80% protein), 45 g/L rice protein concentrate (labeled as80% protein), 3.6 g/l maltodextrin, 1.8 g/L carrot powder, 1.8 g/Ldiammonium phosphate, 0.7 g/L magnesium sulfate heptahydrate, 1.5 g/Lanti-foam and 0.6 g/L citric acid. Seed reactor was also prepared in 200L bioreactor with medium volume of 100 L with the following mediumcomponents:pea protein 5 g/l, rice protein 5 g/l, maltodextrin 3.0 g/l,carrot powder 1 g/l, malt extract 3 g/l and 1.25 g/l of anti-foam. Themedium was inoculated with flask process developed the same way as shownin Example 2. The 200 L bioreactor was harvested 40 to 70 hourspost-inoculation. The flasks were harvested 11 days post-inoculation.The organism was Lentinula edodes sourced from the Penn State mushroomculture collection.

Once the main fermenter was cooled it was inoculated with the 100 Linoculum from the 200 L fermenter. Fermenter was held at 26° C. Theculture in the 4,000 L vessel was harvested at 48 hours post-inoculationupon successful visible (mycelial pellets) and microscope checks.Material was pasteurized in the bioreactor at 65° C. for 60 minutes.Fermenter was then cooled down and material was harvested in sanitized55 gallon drums and sent to spray drying facility.

Example 8

Sensory Data

Eight protein powders were tested: (a) raw material (3.2 pea); (b) rawmaterial (pea); (c) raw material (rice); (d) raw material (rice); (e)myceliated material 3; (f) myceliated material 4; (g) myceliatedmaterial 4.2; and (h) myceliated material 3.2. Each protein powder wastested at 7% in water. Trained descriptive panelists used a consensusdescriptive analysis technique to develop the language, ballot and rateprofiles of the protein powders. The aroma language was as follows:

The raw pea product prior to myceliation has a pea aroma with no rice ormushroom aroma. The rice samples prior to myceliation have rice aromawith no pea or mushroom aroma. After myceliation, these samples havemushroom aroma and no pea or rice aroma, respectively. There is alsoincreased umami flavor in the myceliated samples.

Example 9

Eight (8) 1 L baffled DeLong Erlenmeyer flasks were filled with 0.500 Lof the following 8 different media, after the manner of Example 1, seeTable 2:

TABLE 2 Component Medium 1 Medium 2 Medium 3 Medium 4 Medium 5 Medium 6Medium 7 Medium 8 Pea protein 1 54 54 49.5 54 54 54 0 54 (g/L) Chickpeapowder 36 36 22.5 36 36 36 36 36 (g/L) Rice protein (g/L) 0 0 18 0 0 0 00 Magnesium 0.72 0.72 0.72 0.72 0.72 0.72 0.72 0.72 sulfate (g/L)Diammonium 1.8 1.8 1.8 1.8 1.8 1.8 1.8 1.8 phosphate (g/L) Citric acid(g/L) 1.5 1.5 1.5 1.5 0.6 0.9 1.5 1.5 Carrot powder 1.8 1.8 1.8 1.8 1.81.8 0 1.8 (g/L) Anti-foam 1 (g/L) 1.25 0 1.25 1.25 1.25 1.25 1.25 1.25(organic polymer based) Pea protein 2 0 0.1 0 0 0 0 54 0 (g/L) Anti-foam2 (g/L) 0 0.1 0 0 0 0 0 0 (silicone based) Vegetable juice 0 0 0 0 0 0 50 (mL/L)

The flasks were covered with a stainless-steel cap and steam sterilized.The flasks were carefully transferred to a clean HEPA laminar flow hoodwhere they cooled for 4 hours and each were inoculated with 5% of 10-dayold submerged Lentinula edodes. All 8 flasks were placed on a shakertable at 150 rpm with a swing radius of 1″ at room temperature andallowed to incubate for 3 days. Plating aliquots of each sample on LBand petri film showed no contamination in any flask. The pH changesduring processing is essentially the same (within the margin of error ofthe pH meter).

Top performing recipes in sensory from these 8 media were media 5 and 7.Bitterness and sourness were evaluated and these two media showed thebest results, although all media exhibited reduced undesirable flavorsand reduced aromas, such as reduced beany aroma, pea aroma, or ricearoma and reduced beany taste, pea taste, rice taste, and bitter taste.The sensory evaluation included 15 tasters, all tasting double-blind,randomized samples and providing a descriptive analysis. These recipeswere further evaluated for strain screening work as described in Example10.

Example 10

Eight (8) 1 L baffled DeLong Erlenmeyer flasks were filled with 0.500 Lof medium consisting of the 2 best medium as described in example 11 (4flasks for each medium). These two media were inoculated with fourdifferent species: Lentinula edodes, Boletus edulis, Pleurotussalmoneostramineus and Morchella esculenta. See Table 3.

TABLE 3 Component Medium 1 Medium 2 Pea protein 1 (g/L) 54 0 Chickpeapowder (g/L) 36 36 Magnesium sulfate (g/L) 0.72 0.72 Diammoniumphosphate 1.8 1.8 (g/L) Citric Acid (g/L) 0.6 1.5 Carrot powder (g/L)1.8 1.8 Pea protein 2 (g/L) 0 54 Anti-foam 2 (g/L) 0.1 0.1

The flasks were covered with a stainless-steel cap and sterilized in anautoclave. The flasks were carefully transferred to a clean HEPA laminarflow hood where they cooled for 4 hours and inoculated with 5% of 10-dayold submerged aliquots of each species. All 8 flasks were placed on ashaker table at 150 rpm with a swing radius of 1″ at room temperatureand incubated for 3 days at which point pH was measured and isessentially the same.

Plating aliquots of each sample on petri film showed no contamination inany flask. Bitterness and sourness were evaluated and these two mediashowed the best results, although all media exhibited such as reducedbeany aroma, pea aroma, or rice aroma and reduced beany taste, peataste, rice taste, and bitter taste. The results that were obtainedshowed that Boletus edulis performed better than other species for lowersourness and bitterness. M. esculenta did not perform well.

Example 11

A 7 L bioreactor was filled with 4.5 L of a medium consisting of themedium as described in following table (see Table 4):

TABLE 4 Component Medium 1 Medium 2 Medium 3 Pea protein 1 (g/L) 45 4558.5 Rice Protein (g/L) 45 45 31.5 Anti-foam 2 (g/L) 1.25 1.25 1.25

In this experiment, excipients other than an anti-foam were omitted fromthe fermentation medium, and only rice protein, pea protein, andanti-foam were used as the medium. In previous examples, excipients suchas magnesium sulfate, diammonium phosphate (which functions at least inpart as a buffer), citric acid, carrot powder, were used and wereomitted here. It was theorized that omission of these excipients willencourage the culture to convert protein metabolically and notproliferate. Open ports on the bioreactor were wrapped in foil and thevessel was subsequently sterilized in an autoclave. The bioreactors werecarefully transferred to a clean bench in a cleanroom, setup and cooledfor 4-6 hours. The bioreactor was inoculated with 5%, 10% and 7.5% ofinoculant of L. edodes from a 12-day old flask. Fermentation for thesebatches was completed in 44 hours, 24 hours and 30 hours respectivelyfor medium 1, medium 2 and medium 3. A microscope check was done toensure the presence of mycelium (mycelial pellets were visible by thenaked eye) and the culture was plated on LB media to ascertain theextent of any bacterial contamination and none was observed. Thesecultures were pasteurized for 60 minutes at 65° C.

Microscopic examination of these different inoculum and protein sampleswas done and it suggested growth even for medium 1 at 24 hoursfermentation.

Bitterness and sourness were evaluated and all media exhibited reducedbeany aroma, pea aroma, or rice aroma and reduced beany taste, peataste, rice taste, and bitter taste.

Example 12

Vegan Protein Bites

The following materials are mixed and formed into a bar: PURETASTEprotein (70% protein/100 g product; mixture of 65% pea protein and 35%rice protein, made by processes of the invention at Example 11) at 11.4g; tapioca syrup, 21 g; sugar, granulated white; 3.6 g; honey, clover,3.6 g; canola oil, 3 g; almond flour (6 g protein/28 g flour), 9 g;peanut butter (8 g protein/32 g), 3 g; vanilla extract, 0.6 g, andflavoring (Pomegranate), 0.09 g. PURETASTE is used at 20.8%. UsingPURETASTE, beany and bitter off-flavors do not occur in the bar and thebar has good appearance, flavor, and mouth-feel.

Yogurt

The following materials are mixed: almond milk, 68.7%; cashew milk,21.9%; coconut cream, 3.35%; PURETASTE (70% protein/100 g product;mixture of 65% pea protein and 35% rice protein), 4.75%; dextrose,1.15%; locust bean gum, 0.05%; pectin, 0.05%; and cultures, 0.02%; andyogurt is produced using conventional processes. Using PURETASTE, beanyand bitter off-flavors do not occur in the ice cream, and the bar hasgood appearance, flavor, and mouth-feel.

Ice Cream

The following materials are mixed: water, 45.475%; coconut cream (43.7%fat), 32.015%; sugar, 17%; PURETAS (70% protein/100 g product; mixtureof 65% pea protein and 35% rice protein), 4.5%; TIC GUMS Natural IC CL,0.6%; sunflower lecithin, 0.2% and sea salt, 0.21%. When tasted, the icecream provided complete protein dairy alternative dessert that containsno vegetable off-notes from the protein source.

Example 13

Summary of Crisps and Scoops:

PureTaste® protein (PTP) (made as described in Example 11) wereprocessed to form extruded crisps ranging in protein content withvarying degrees of expansion, color and texture.

Compared to extruded soy crisps which had beany/chalky notes, PureTaste®protein crisps flavor ranged from light malted, toasted, grainy notes tovery light earthy notes at elevated levels of PureTaste® protein.High-protein (62% Protein) PureTaste® protein extruded scoops producedwere tan in color, toasted brown notes, latent slight earthy notes.

PureTaste® Crisps (PT Crisps)

Trials were conducted with Wenger X57 Twin Screw Extruder. 4 trials wereconducted for production of PTP crisps as shown below:

Crisp: Control (Soy Protein Isolate 84.25%+Tapioca Starch 15%+Calciumcarbonate 0.75%), protein content 76%. Density was 325 g/L, color wasoff-white, texture was crispy/crunchy, flavor was light soy notes.

Crisp: PTP (84.25%)+15% Tapioca Starch+0.75% Calcium Carbonate (proteincontent 63%). Density is 269 g/L, color is tan, texture wascrispy/crunchy, flavor was toasted brown notes. PTP=PURETASTE protein,as made in Example 11.

Crisp: PTP (90%)+10% Tapioca Starch (protein content 68%). Density was222 g/L, color was tan, texture was crispy/crunchy, flavor was toasted.

Crisp: PTP (85%)+5% Tapioca Starch+10% pea protein isolate (proteincontent 72%). Density was 261 g/L, color was dark tan, texture wascrispy/crunchy, flavor was toasted.

Crisp: PTP (90%)+5% pea protein isolate+5% pea starch (protein content72%). Density was 211 g/L, color was dark tan, texture wascrispy/crunchy, flavor was toasted.

Crisp: PTP (70%)+30% Tapioca Starch. Protein content was 75%. Densitywas 262 g/L, color was dark tan, texture was crispy/crunchy, flavor wastoasted.

Scoop: PTP (79.25%)+20% Tapioca Starch. Protein content was 60%. Densitywas 109 g/L, color was brown, texture was light, crunchy, flavor wastoasted.

Scoop: PTP (85%)+5% Tapioca Starch+10% pea protein isolate. Proteincontent was 72%. There was only limited expansion, color was brown,texture was harder, crunchy, flavor was toasted.

Processing Conditions:

A. Raw material information. Dry recipe density 490-530 kg/m³; dryrecipe rate 140-160 kg/hr; feeder speed 30-45 rpm; live bin weight 20-25kg.

Preconditioner information. Small side speed 750-800 rpm; large sidespeed, 100-150 rpm; mixing intensity 70-90%; cylinder steam 0.05-0.2kg/hr; cylinder water 40-50 kg/hr; cylinder discharge temp, 25-35° C.

Extruder information. Extruder speed, 300-500 rpm. The zone 1 temp was50-70° C. (SP) and 50-70° C.(PV), zone 2 temp was 90-110° C.(SP) and90-110° C. (PV); zone 3 temp was 110-130° C.(SP) and 110-130° C.(PV);zone 4 temp was 110-130° C.(SP) and 110-130° C.(PV); zone 5 temp was80-100° C.(SP). The die pressure was 1500-2500 kPA.

Dryer information: zone 1 temperature 140-160° C.; zone 2 temperature,60-85° C., zone 3 temperature, 70-90° C.; retention time, Pass 1, 8-15minutes; Retention time, Pass 2, 8-15 minutes, exhaust 1 temperature110-130° C.; exhaust 2 temperature 70-85° C.

Final product information. Extruder discharge density 200-270 kg/m³;moisture content 3%.

Results: Successfully produced PTP crisps & scoops with lighter densitythan control, varying in color content, malted, toasted notes with verylight latent after notes. PTP Crisps ranged in protein content. Scoopsprotein content was about 62%. At 72% protein level the scoops hadlimited expansion.

Example 14

Summary of Texturized Protein

PureTaste® protein (PT) in combination with starch, wheat gluten, peaprotein and mineral salts can be processed to produce texturizedvegetable protein with final protein content ranging from 60% to 77%,with varying degrees of expansion volume, color and flavor. Flavor wasacceptable with malted, grainy and latent bitter notes. Successfullyproduced texturized PT protein with good to medium hydration capability,soft and harder texture.

Using Wenger X57 Twin Screw extruder; PureTaste Protein used was a 65%pea protein concentrate, 35% rice protein concentrate blend. (PT) incombination with starch, wheat gluten, pea protein and mineral salts canbe processed to produce texturized vegetable protein with final proteincontent ranging from 60% to 77%, with varying degrees of expansionvolume, color and flavor. Flavor was acceptable with malted, grainy andlatent bitter notes. The texturized PT protein had good to mediumhydration capability, soft and harder texture. Procedure used a modifiedextruder setting, with the addition of a 10-inch spacer or a 12-inchspacer with a Teflon nozzle (Venturi system) additional barrel coated onthe inside with Teflon, followed by a narrow exit through a Tefloncoated die. This set up provided successful texturization.

Test 1. Fine flours of PureTaste® protein, 60%, vital wheat gluten 20%,tapioca starch 20%, were mixed with water to result in 25 to 30% watercontent. Density was 200 kg/m³. After extrusion, the texturized pieceswere in chunk form. The extruded material was a white/tan color and wascrunchy when dried. When rehydrated, the extruded material hadacceptable chewable characteristics, and was softer (not as chewable)compared to a control prepared with soy and had a clean, non-beanyflavor and aroma.

Test 2: Fine flours of pea protein, 100%. Density was 98 kg/m³. Afterextrusion, the texturized pieces were in chunk form. The extrudedmaterial was a medium tan color and was crunchy when dried. Whenrehydrated, the extruded material was as chewy compared to a controlprepared with soy and had a light pea flavor.

Test 3: Fine flours of pea protein isolate, 100%. Density was 141 kg/m³.After extrusion, the texturized pieces were in chunk form. The extrudedmaterial was a medium tan color and was crunchy when dried. Whenrehydrated, the extruded material was as chewy compared to a controlprepared with soy and had a light pea flavor.

Test 4: Fine flours of pea flour, 100%. Density was 113 kg/m³. Afterextrusion, the texturized pieces were in chunk form. The extrudedmaterial was a medium tan color and was crunchy when dried. Whenrehydrated, the extruded material was as chewy compared to a controlprepared with soy and had a light pea flavor.

Test 5: Fine flours of PureTaste® protein, 100%. Density was heavy.After extrusion, the texturized pieces were in crumble form. Theextruded material was a medium tan color and had a clean flavor.

Test 6, Fine flours of PureTaste® protein, 50% and pea protein isolate50% were mixed with water to result in 30% water content. Density was280 kg/m³. Following extrusion, the texturized pieces were in shredsform. The extruded material was a light to medium tan color and wascrunchy when dried. When rehydrated, the extruded material hadacceptable chewable characteristics, and was softer (not as firm)compared to a control prepared with soy and has a clean, non-beanyflavor and aroma.

Test 7. PureTaste® protein 50%, vital wheat gluten 50%, results indensity of 313 kg/m³, protein content of 75%, consistency of chunks.When rehydrated, the extruded material had acceptable chewablecharacteristics, and was almost identical in chewiness and firmnesscompared to a control prepared with soy (but softer) and has a clean,non-beany flavor and aroma.

Test 8. PureTaste® protein 50%, vital wheat gluten 40%, tapioca starch10%, results in density of 204 kg/m³, protein content of 68%,consistency of chunks. When rehydrated, the extruded material hadacceptable chewable characteristics, and was almost identical inchewiness and firmness compared to a control prepared with soy (butsofter) and has a clean, non-beany flavor and aroma.

Test 9. PureTaste® protein 60%, vital wheat gluten 20%, tapioca starch20%, results in density of 177 kg/m³, protein content of 60%,consistency of chunks. When rehydrated, the extruded material hadacceptable chewable characteristics, and was almost identical inchewiness and firmness compared to a control prepared with soy (butsofter) and has a clean, non-beany flavor and aroma.

Test 10. PureTaste® protein 50%, vital wheat gluten 35%, tapioca starch15%, results in density of 179 kg/m³, protein content of 64%,consistency of chunks. When rehydrated, the extruded material hadacceptable chewable characteristics, and was almost identical inchewiness and firmness compared to a control prepared with soy (butsofter) and has a clean, non-beany flavor and aroma.

Test 11. PureTaste® protein 25%, pea protein isolate, results in densityof 132 kg/m³, protein content of 79%, consistency of chunks. Whenrehydrated, the extruded material had acceptable chewablecharacteristics, and was almost identical in chewiness and firmnesscompared to a control prepared with soy (but softer) and has a clean,non-beany flavor and aroma.

Test 12. PureTaste® protein 50%, pea protein 20%, vital wheat gluten20%, pea starch 8.5% (along with 1% calcium chloride, 0.2% sodiummetabisulfite and 0.3% trisodium phosphate) had a density of 215 kg/m³and a protein content of 69%. When rehydrated, the extruded material hadacceptable chewable characteristics, and was almost identical inchewiness and firmness compared to a control prepared with soy (butsofter) and has a clean, non-beany flavor and aroma.

Test 13. PureTaste® protein 60%, vital wheat gluten 20%, tapioca starch20% had a density of 200 kg/m³, and a protein content of 60%. Whenrehydrated, the extruded material had acceptable chewablecharacteristics, and was almost identical in chewiness and firmnesscompared to a control prepared with soy (but softer) and has a clean,non-beany flavor and aroma.

Test 14. PureTaste® protein 25.75%, vital wheat gluten 33%, tapiocastarch 5%, pea protein 15%, pea fiber 20%, with trisodium phosphate0.5%, results in material that when rehydrated, the extruded materialhad acceptable chewable characteristics, and was almost identical inchewiness and firmness compared to a control prepared with soy (butsofter) and had a clean, non-beany flavor and aroma.

Test 15. PureTaste® protein 32.92%, vital wheat gluten 16.46%, tapiocastarch 19.75%, pea protein 29.62%, with trisodium phosphate 0.5%,results in material that when rehydrated, the extruded material hadacceptable chewable characteristics, and was almost identical inchewiness and firmness compared to a control prepared with soy (butsofter) and had a clean, non-beany flavor and aroma.

Processing Conditions:

A. Raw material information. Dry recipe density 550-600 kg/m³; dryrecipe rate 90-110 kg/hr; feeder speed 20-25 rpm; live bin weight 20-35kg.

Preconditioner information. Small side speed 150-800 rpm; large sidespeed, 100-130 rpm; mixing intensity 70-90%; cylinder steam 1.8-2.5kg/hr; cylinder water 25-35 kg/hr; cylinder discharge temp, 40-50° C.

Extruder information. Extruder speed, 300-700 rpm; The zone 1 temp was50-70° C. (SP) and 50-70° C.(PV), zone 2 temp was 90-110° C.(SP) and90-110° C. (PV); zone 3 temp was 120-140° C.(SP) and 120-130° C.(PV);zone 4 temp was 125-135° C.(SP) and 115-135° C.(PV); zone 5 temp was80-100° C.(SP). The die pressure was 2000-2300 kPA.

Dryer information: zone 1 temperature 120-130° C.; zone 2 temperature,80-100° C., zone 3 temperature, 80-100° C.; retention time, Pass 1, 8-12minutes; Retention time, Pass 2, 7-10 minutes, exhaust 1 temperature100-130° C.; exhaust 2 temperature 80-100° C.

Results: Successfully produced PTP texturized protein.

Other tests resulted in crumbles or chunks that disintegrated in waterupon rehydration. This includes formulations (a) 50% PTP, 25% VWG, 5%pea fiber, 8.5% pea starch, 10% pea protein 55%, (along with 1% calciumchloride, 0.2% sodium metabisulfite and 0.3% trisodium phosphate); (b)75% PTP, 10% tapioca starch, 13.5 pea protein 55%, (1% calcium chloride,0.2% sodium metabisulfite and 0.3% trisodium phosphate); (c) 55% PTP,20% VWG, 5% tapioca starch, 19.7% pea protein 55% protein with onlytrisodium phosphate; (d) 78.5% PTP, 10% tapioca starch, 10% pea protein80%, (along with 1% calcium chloride, 0.2% sodium metabisulfite and 0.3%trisodium phosphate); (e) 60% PTP, 20% VWG, 8.5% pea starch, 10% peaprotein 55% (along with 1% calcium chloride, 0.2% sodium metabisulfiteand 0.3% trisodium phosphate); (f) 75% PTP, 10% VWG, 5% tapioca starch,8.5% pea protein 55% (along with 1% calcium chloride, 0.2% sodiummetabisulfite and 0.3% trisodium phosphate); (g) 60% PTP, 8.5% tapiocastarch, 30% pea protein 80%, (along with 1% calcium chloride, 0.2%sodium metabisulfite and 0.3% trisodium phosphate); (h) 60% PTP, 30%VWG, 10% tapioca starch, no additional excipients; (i) 60% PTP, 20%tapioca starch, 20% AGT protein 56; no additional excipients (j) 60%PTP; 25% VWG, 15% pea protein 55%, no additional excipients.

Example 15

Texturized protein with pea fiber. 50-45-5. PureTaste® protein 50%, peaprotein isolate 45%, pea fiber 5%, results in material that whenrehydrated, the extruded material had acceptable chewablecharacteristics, and was almost identical in chewiness and firmnesscompared to a control prepared with soy (but softer) and had a clean,non-beany flavor and aroma. Product dimensions were length, 12.0-16.0mm, width 5.0 to 6.0 mm, and thickness 3.0 to 5.0 mm, with granulationof 7/16″ or 0.438″ or 11.12 mm>10%; ¼″ or 0.25 in or 6.35 mm 30-50%; and6 mesh or 0.132″ or 3.35 mm 50% to 30% of total.

Texturized protein with pea fiber. 70-25-5. PureTaste® protein 70%, peaprotein isolate 25%, pea fiber 5%, results in material that whenrehydrated, the extruded material had acceptable chewablecharacteristics, and was almost identical in chewiness and firmnesscompared to a control prepared with soy (but softer) and had a clean,non-beany flavor and aroma. Product dimensions were length, 12.0-16.0mm, width 5.0 to 6.0 mm, and thickness 3.0 to 5.0 mm, with granulationof 7/16″ or 0.438″ or 11.12 mm>10%; ¼″ or 0.25 in or 6.35 mm 30-50%; and6 mesh or 0.132″ or 3.35 mm 50% to 30% of total.

Processing Conditions:

A. Flour mix feed rate 13 lbs at 17 lbs/min; preconditioner, wateraddition in the range of 2.0 to 4.0 lbs/min; steam incorporation at 0.4lb to 0.6 lbs/min; extruder; water addition ranged from 0.75 lbs to 1.0lbs/min, extruder RPM is 350 to 400 rpm; extruder temperatures: zone 1,155° F. to 165° F.; zone 2, 265° F. to 280° F.; zone 3, 285° F. to 295°F.; zone 4, 280° F. to 300° F.; pressures in the range of 800 to 1000psi, dryer temperatures 250° F., 110° F.; product density 0.12 to 0.22gm/cc; product moisture 5% to 7%.

Results: Successfully produced PTP texturized protein. Improvedtexturization with pea fiber achieved.

Properties of PureTaste™ Texturized Protein.

Compared with texturized protein using pea protein only. PureTaste™texturized protein (both formulations with pea fiber), by sensorytesting, have a harder bite, more spring, resists becoming a paste uponcompression. The flavor is less pea and less earthy. PureTaste™texturized protein, compared with soy texturized protein. PureTaste™texturized protein (both formulations with pea fiber), by sensorytesting, have more spring and the particle size is larger; increasedsavory flavor and less sweet flavor, and had similar water adsorption.See Table 5 and 6.

Attributes;

TABLE 5 50% 70% Attribute Definition Soy Pea PTP PTP Springiness Thedegree and rate to which 7 7 9 10.5 the sample returns to its originalshape. Hardness The force required to obtain 5.5 6 7 9.5 deformation.Cohesive- The amount of sample that 9.5 8 8 8 ness deforms rather thanshears/cuts. Moisture The amount of moisture 4.5 2 5 4 Release(juice/oil/water) perceived in (Juiciness) the mouth. Moisture of Theinnate moisture of a 5 5 4 4.5 Mass sample before it has been altered.Density The compactness of the 10 10 10 11 sample cross sectionCohesive- The degree to which a chewed 6 7 7 7.5 ness of sample holdstogether in a Mass mass. Toothpack The amount of product 5.5 6 4 5adhering to the teeth after mastication of the sample.

Flavor attributes. Compared with soy texturized protein and peatexturized protein, the flavors of the formulations in this Example hadan improvement over pea texturized protein in that both formulationslacked a beany flavor (less pea flavor) and had increased cereal andmalty notes, and less metallic and chalky flavor notes. Bitterness forthe PureTaste was reduced compared to pea texturized protein.

Texturized Protein Water Adsorption Study;

TABLE 6 Ratio of water adsorbed to water Weight at Initial water Volumeof input Time to full full required for water (vol. of hydrationhydration hydration adsorbed adsorption/initial (min) (g)* (ml) (ml)water) Soy Texturized 10 72 85 55 0.65 Protein Pea Texturized 25 87 9068 0.76 Protein PureTaste 725 25 59 100 42 0.42 PureTaste 545 15 76 22057 0.26

Example 16

Meat analog patty. Using the successful PT texturized protein materialsmade in Example 15, a meat analogue patty was made. The ingredients inwt percent were PureTaste texturized protein 10.42%, PureTaste Protein6.01%, Pea Protein texturized protein 8.42%, Vital Wheat Gluten 7.62%,Methylcellulose 2.00%, Beef Flavor 2.20%, Grill Flavor, 2.61%, ChickenFlavor 1.80%, Beet Powder 0.70%, Unrefined Coconut Oil 2.00%, brownflavor 0.1%, water 56.11%. After grilling the patty, the tasters agreedthat the patty had good flavor and texture.

Meat extender patty. Using the successful PT texturized proteinmaterials made in Example 14, a meat extender patty was made. Theingredients in wt percent were PureTaste texturized protein 3.27%,PureTaste Protein 2.84%, Pea Protein texturized protein 2.98%, VitalWheat Gluten 1.99%, Methylcellulose 0.71%, Beef Flavor 1.85%, BeetPowder 0.28%, water 11.08%, and ground beef 80:20 (protein:fat), 75%.After grilling the patty, the tasters agreed that the patty had goodflavor and texture.

Meat extender patty. Using the successful PT texturized proteinmaterials made in Example 14, to a beef mixture, PT texturized proteinwas added at amounts up to 2% and water was increased up to 7% over thecontrol's 8.5%. Compared with control, the additional moisture was heldin the product, indicating a cost savings obtained with PT texturizedprotein. Taste changes compared with a pure beef patty were very small.

Pork meatballs and pork sausage patty. PT was added as a meat extenderto a pork meatball and pork sausage at 1.5%, 2.5%, and 5%. A control hadsoy added at 1.5%. The overall taste was enhanced relative to controlwith 5% being the best taste. PureTaste was able to hold increasedamounts of water relative to control (with the product more juicy andlowering cost, with the product less dense and more desirable.) Thecontrol had 14% moisture and it was increased to 16% using PURETASTEprotein.

Meatless meatballs. PT texturized protein (Example 15; 5-45-5formulation) was added as the main ingredient to a vegetarian meatball(other than water for rehydration). The other ingredients includedPureTaste™ protein powder, canola oil, coconut oil, contains less than2% each of the following: rice, methylcellulose, natural flavors, beetpowder, yeast extract, sea salt, modified food starch, garlic powder,onion powder, black pepper, beta carotene.

Meatless tacos. PT texturized protein (Example 15; 5-45-5 formulation)was added as the main ingredient to a vegetarian meatball (other thanwater for rehydration). The other ingredients included canola oil, vitalwheat gluten, methylcellulose, coconut oil, natural flavors, spices(paprika, salt, chili pepper, oregano, corn starch, dried onion, driedgarlic).

High fat meat analog. Ingredients: water for hydration, PT texturizedprotein (made per Example 14), coconut fat, coconut oil, flavors,methylcellulose, 2% or less of spices, beet powder, salt. 24 g proteinper serving, 18 g fat per serving, 2 g carbohydrate per serving.

Beef frankfurter. Using the successful PT texturized protein materialsmade in Example 14, a beef frankfurter is made. The ingredients in wtpercent were beef trim (10% fat) at 26%, pork trim fat at 20%, PureTastetexturized protein 4.0%, PureTaste Protein 5.0%, salt at 2.0%, phosphate0.4%, sodium nitrate at 0.1% (140 ppm), sodium ascorbate at 0.4%,dextrose at 1.0%, corn syrup solids at 2.0%, sodium lactate at 3.0%,flavors and spices at 0.1%, and water 36%. Upon grilling thefrankfurter, the tasters agree that the frankfurter has good flavor andtexture.

Pork sausage. Using the PT texturized protein materials made in Example14, a pork sausage is made. The ingredients in wt percent were pork 85%lean at 70%, PureTaste texturized protein 10.0%, PureTaste Protein 7.5%,water at 10%, and seasoning at 2.5%. Upon cooking the sausage, thetasters agree that the sausage has good flavor and texture.

Although the examples above show the use of twin-screw extruder, itshould be understood that extrusion processes are very diverse andmanufacturing of extruded compositions as disclosed herein containingproduct can be prepared via use of any acceptable model of type foodprocessing extruder, both with single screw or with twin screw types.

Example 17

Non-dairy, beverage, bakery, and pasta. All materials made fromPureTaste™ powder made as disclosed in Example 11.

Red lentil high protein, gluten free pasta. 20 g protein per serving.Ingredients: eggs, PureTaste™ powder, red lentil flour. Pasta mixturewas hydrated and formed into pasta using art methods for making pasta.

Red lentil and garbanzo high protein, gluten free pasta. 20 g proteinper serving. Ingredients: eggs, PureTaste™ powder, garbanzo bean flour,red lentil flour. Pasta mixture was hydrated and formed into pasta usingart methods for making pasta.

Red lentil high protein, gluten free pasta. 15 g protein per serving.Ingredients: red lentil flour, eggs, PureTaste™ powder, olive oil. Pastamixture was hydrated and formed into pasta using art methods for makingpasta.

Semolina high protein pasta. 10 g protein per serving. Ingredients:Enriched durum flour (Durum wheat, iron, niacin, thiamine, riboflavin,folic acid), eggs, PureTaste™ powder, olive oil. Pasta mixture washydrated and formed into pasta using art methods for making pasta.

Pancakes. 5 g protein per serving. Skim milk, enriched bleached wheatflour (flour, niacin, iron thiamine mononitrate, riboflavin, enzymes,folic acid), PureTaste™ powder, eggs, butter (sweet cream, naturalflavoring), baking powder, sugar, vanilla extract, salt. Pancakes weremade by art methods using the above ingredients.

Low salt crackers. 2 g protein per serving. Enriched bleached wheatflour (flour, niacin, iron, thiamine mononitrate, riboflavin, enzymes,folic acid), water, PureTaste™ protein, canola oil, sugar, bakingpowder, baking soda, salt. Crackers were made by art methods using theabove ingredients.

Vegan protein muffins. 7 g protein per serving. Almond milk, enrichedbleached wheat flour (flour, niacin, iron, thiamine mononitrate,riboflavin, enzymes, folic acid), sugar, PureTaste™ protein, canola oil,less than 2% of the following: baking powder, apple cider vinegar,cellulose and xanthan gums, vanilla extract, cinnamon, salt, nutmeg.Muffins were made by art methods using the above ingredients.

High protein muffins. 7 g protein per serving. Egg whites, cane sugar,flour (bleached wheat flour, malted barley flour, niacin, iron, thiaminemononitrate, riboflavin, enzymes, folic acid), dark chocolate morsels(unsweetened chocolate, cane sugar), eggs, PureTaste™ protein, canolaoil, vanilla extract, vital wheat gluten, nonfat dry milk, bakingpowder, sunflower lecithin, salt, xanthan gum. Muffins were made by artmethods using the above ingredients.

High protein donuts. 19 g protein per serving. Sugar, flour (bleachedwheat flour, malted barley flour, niacin, iron, thiamine mononitrate,riboflavin, enzymes, folic acid), PureTaste™ protein powder, unsweetenedalmond milk (water, almonds), eggs, unsalted butter, vegetable oil,contains 2% or less of: vanilla extract, baking powder, salt, bakingsoda, nutmeg. Donuts were made by art methods using the aboveingredients.

High protein bread. 10 g protein per serving. Bread flour (unbleachedhard red wheat flour, malted barley flour), water, PureTaste™ protein,cane sugar, nonfat dry milk, salt, vegetable shortening (soybean oil,fully hydrogenated palm oil, palm oil, mono and diglycerides, TBHQ andcitric acid), yeast (yeast, sorbitan monostearate, ascorbic acid), wheatgluten, cellulose gum, xanthan gum. Bread was made by art methods usingthe above ingredients.

Vegan gluten-free brownies. 10 g protein per serving. Almondmilk,PureTaste™ protein, sugar, applesauce, margarine (vegetable oil blend[palm, canola and olive oils], water, annatto extract, lactic acid,natural flavor, pea protein, sunflower lecithin), cocoa powder, glutenfree powder (garbanzo bean flour, potato starch, tapioca flour, whitesorghum flour, fava bean flour), chocolate morsels (sugar, chocolate,cocoa butter), peanut butter, less than 2% of the following: bakingpowder, cellulose and xanthan gums, vanilla extract, cinnamon. Brownieswere made by art methods using the above ingredients.

Vegan drinkable yogurt. 6 g protein per serving. Water, oat base (water,rolled oats), PureTaste™ powder dextrose, sunflower lecithin, locustbean gum, pectin, live and active cultures. Mixture was treated by artmethods to create a suspension/emulsion.

Chocolate “milk”. PureTaste™ powder was added as the main ingredient toa chocolate “milk” (other than water for rehydration). The otheringredients included cane sugar, coconut oil, canola oil, cocoa(processed with alkali), natural flavors, gum acacia, gellan gum, seasalt, sunflower lecithin. Mixture was treated by art methods to create asuspension/emulsion.

Vanilla yogurt. PureTaste™ powder was added as the main ingredient(other than water for rehydration). The other ingredients included oatconcentrate (water, rolled oats), cane sugar, coconut oil, dextrose,locust bean gum, pectin, vanilla, lemon juice concentrate, sunflowerlecithin, live and active cultures. Mixture was treated by art methodsto create a suspension/emulsion.

Strawberry yogurt. PureTaste™ powder was added as the main ingredient(other than water for rehydration). The other ingredients included oatconcentrate (water, rolled oats), cane sugar, coconut oil, strawberries,dextrose, pectin, natural flavor, locust bean gum, fruit and vegetablejuice (for color), lemon juice concentrate, sunflower lecithin, live andactive cultures. Mixture was treated by art methods to create asuspension/emulsion.

Coconut yogurt. PureTaste™ powder was added as the main ingredient(other than water for rehydration). The other ingredients included oatconcentrate (water, rolled oats), cane sugar, coconut oil, coconutcream, coconut, dextrose, pectin, locust bean gum, lemon juiceconcentrate, natural flavor, sunflower lecithin, live and activecultures. Mixture was treated by art methods to create asuspension/emulsion.

Vegan Greek yogurt. PureTaste™ powder was added as the main ingredient(other than water for rehydration). The other ingredients included oatconcentrate (water, rolled oats), cane sugar, coconut oil, dextrose,locust bean gum, pectin, sunflower lecithin, live and active cultures.Mixture was treated by art methods to create a suspension/emulsion.

Vegan chocolate pudding. 5 g protein per serving. Coconut milk (water,coconut cream), can sugar, PureTaste™ powder, high fat cocoa, gumacacia, guar gum, locust bean gum, natural flavor, sea salt, sunflowerlecithin. Mixture was treated by art methods to create asuspension/emulsion.

Protein Shake powder (chocolate). PureTaste™ powder, sugar, cocoa(processed with alkali), natural flavors, stabilizer blend (guar gum,gum acacia, xanthan gum), gum Arabic, sunflower oil, salt, sunflowerlecithin, steviol glycosides.

Protein Shake powder (vanilla). PureTaste™ powder, sugar, naturalflavors, stabilizer blend (guar gum, gum acacia, xanthan gum), gumArabic, sunflower oil, salt, sunflower lecithin, steviol glycosides.

Nutritional shake (unsweetened and unflavored): water, PureTaste™powder, canola oil, gum acacia, gellan gum, sunflower lecithin.

Whole “milk”. PureTaste™ powder was added as the main ingredient to awhole “milk” (other than water for rehydration). The other ingredientsincluded coconut oil, cane sugar, canola oil, gum acacia, gellan gum,sunflower lecithin. Mixture was treated by art methods to create asuspension/emulsion.

Vegan ice cream. 5 g protein per serving. Ingredients: coconut milk(water, coconut cream), cane sugar, PureTaste™ protein powder, gumacacia, guar gum, locust bean gum, natural flavor, sea salt, sunflowerlecithin. Ice cream was made by methods known in the art.

Vegan chocolate ice cream. 6 g protein per serving. Ingredients: coconutmilk (water, coconut cream), cane sugar, PureTaste™ protein powder, highfat cocoa, gum acacia, guar gum, locust bean gum, natural flavor, seasalt, sunflower lecithin. Ice cream was made by methods known in theart.

Example 18

Reaction flavor. To 165 g distilled deionized water in a glass beaker,220 g (dry weight) material as prepared in Example 11 was added. To thismixture was added 55 g of a reducing carbohydrate source, caustic 6.5 gof 1 N HCl, under vigorous stirring to form a homogenous suspension. Thefree flowing slurry was then processed in in a closed, conventional,double-jacketed glass reactor (volume 1 L) equipped with an anchorstirrer and a temperature sensor. After establishing a suspension of thestarting materials at 50° C., the slurry was heated to 115° C. within 70minutes. After reaching 115° C., the slurry was kept at this temperaturefor 65 minutes (pressure buildup is observed, 1.5 bar). After thereaction, the batch was cooled down to 50° C. within 40 minutes. Theresulting liquid was blended with an amount of carrier and spray driedon a Niro Minor type spray tower. This spray dried material wasevaluated by trained panelists and was determined to have an intense andcomplex flavor reminiscent of flavors obtained by cooking techniques.

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The use of the terms “a” and “an” and “the” and “at least one” andsimilar referents in the context of describing the invention (especiallyin the context of the following claims) are to be construed to coverboth the singular and the plural, unless otherwise indicated herein orclearly contradicted by context. The use of the term “at least one”followed by a list of one or more items (for example, “at least one of Aand B”) is to be construed to mean one item selected from the listeditems (A or B) or any combination of two or more of the listed items (Aand B), unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations of those preferred embodiments may become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

1. A method to prepare a food composition, comprising the steps of: (a)providing a myceliated high-protein food product, comprising the stepsof either: (i) providing an aqueous medium comprising a high-proteinmaterial, wherein the aqueous medium comprises at least 50% (w/w)protein on a dry weight basis, wherein the media comprises at least 50g/L protein; inoculating the medium with a fungal culture, and culturingthe medium to produce a myceliated high-protein food product; or, (ii)providing a myceliated high-protein food product, wherein the myceliatedhigh-protein food product is at least 50% (w/w) protein on a dry weightbasis, wherein the myceliated high-protein product is myceliated by anaqueous fungal culture, in a media comprising at least 50 g/L protein inliquid culture; wherein the fungal culture comprises Lentinula edodes,Agaricus spp., Pleurotus spp., Boletus spp., or Laetiporus spp.; andwherein the myceliated high-protein food product is derived from a plantsource and has reduced undesirable flavors and reduced undesirablearomas compared to the high-protein material that is not myceliated; (b)providing an edible material; and (c) mixing the myceliated high proteinfood product and the edible material to form the food composition. 2.The method of claim 1, wherein the Laetiporus spp. is Laetiporussulfureus, wherein the Pleurotus spp. comprises Pleurotus ostreatus,Pleurotus salmoneostramineus (Pleurotus djamor), Pleurotus eryngii, orPleurotus citrinopileatus, and wherein Boletus spp. comprises Boletusedulis and wherein Agaricus spp. comprises Agaricus blazeii, Agaricusbisporus. Agaricus campestris, Agaricus subrufescens, Agaricusbrasiliensis or Agaricus silvaticus
 3. The method of claim 1, whereinthe high-protein material is at least 70% (w/w) protein on a dry weightbasis and is a protein concentrate or protein isolate.
 4. The method ofclaim 1, wherein the plant source comprises pea, rice, or combinationsthereof.
 5. The method of claim 1, wherein the method comprises theculturing step (a)(i) and the culturing step is carried out until thedissolved oxygen in the media reaches between 80% and 90% of thestarting dissolved oxygen and wherein the pH of the fungal culture has achange of less than 0.5 pH units during the processing step.
 6. Themethod of claim 5, wherein the pH of the fungal culture does not changeduring processing.
 7. The method of claim 1, wherein the reducedundesirable flavor is a pea flavor or a bitterness flavor and thereduced undesirable aroma is a beany aroma or a rice aroma.
 8. Themethod of claim 1, further comprising a cooking step and an extrusionstep using an extruder.
 9. The method of claim 8, further comprising apuffing step.
 10. The method of claim 1, wherein the edible materialcomprises a starch, a flour, a grain, a lipid, a colorant, a flavorant,an emulsifier, a sweetener, a vitamin, a mineral, a spice, a fiber, aprotein, nutraceuticals, sterols, isoflavones, lignans, glucosamine, anherbal extract, xanthan, a gum, a hydrocolloid, a starch, apreservative, a legume product, a food particulate, or combinationsthereof.
 11. The method of claim 10, wherein the food particulate isselected from the group consisting of cereal grains, cereal flakes,crisped rice, puffed rice, oats, crisped oats, granola, wheat cereals,protein nuggets, texturized plant protein ingredients, flavored nuggets,cookie pieces, cracker pieces, pretzel pieces, crisps, soy grits, nuts,fruit pieces, corn cereals, seeds, popcorn, yogurt pieces, andcombinations of any thereof.
 12. The method of claim 1, wherein the foodcomposition is selected from the group consisting of dairy alternativeproducts, ready to mix beverages and beverage bases; extruded andextruded/puffed products; sheeted baked goods; meat analogs andextenders; baked goods and baking mixes; granola; and soups/soup bases.13. The method of claim 1, wherein the method additionally comprises (d)adding steam and/or water to the mixture; (e) extruding the mixtureunder heat and pressure to form a textured plant-based protein product,wherein the edible material comprises an additional high proteinmaterial, and wherein the myceliated high-protein food product ispresent at between about 5% and 90% on a dry weight basis compared withthe edible material.
 14. The method of claim 13, wherein the methodfurther comprises providing a starch or a fiber prior to the mixingstep.
 15. The method of claim 13, wherein the plant source is peaprotein and/or rice protein, and wherein the reduced undesirable flavoris a pea flavor or a bitterness flavor and the reduced undesirable aromais a beany aroma or a rice aroma.
 16. The method of claim 13, whereinthe edible material comprises pea protein.
 17. The method of claim 13,wherein the mixture further comprises a starch, a flour, a grain, alipid, a colorant, a flavorant, an emulsifier, a sweetener, a vitamin, amineral, a spice, a fiber, an enzyme, a protein powder, nutraceuticals,sterols, isoflavones, lignans, glucosamine, an herbal extract, xanthan,a gum, a hydrocolloid, a preservative, a legume product, a foodparticulate, or combinations thereof.
 18. The method of claim 13,wherein the mixture comprises about 45% pea protein, 5% pea fiber, andabout 50% myceliated high-protein material, w/w.
 19. The method of claim13, wherein the mixture comprises about 70% myceliated high-proteinmaterial, about 25% pea protein, about 5% pea fiber, w/w.
 20. The methodof claim 1, wherein the method further comprises: (d) processing themixture to form a reaction flavor composition, wherein the ediblematerial is a reaction flavor component capable of facilitating Maillardand/or Strecker reactions.
 21. The method of claim 20, wherein the atleast one reaction flavor component capable of facilitating Maillardand/or Strecker reactions comprises glucose, ribose, fructose, datesyrup, high fructose corn syrup, malted barley, agave syrup, tapiocasyrup, brown rice syrup, calcium carbonate, ascorbic acid, sodiumascorbate, calcium ascorbate, and/or potassium ascorbate, andcombinations thereof, wherein the one or more ingredients capable offacilitating Maillard and/or Strecker reactions comprise approximately0.01% to 6% of the reaction flavor composition; wherein the pH of themixture is in the range of 8 to 9; and wherein processing the mixturecomprises holding the mixture at a temperature of between 80° C. to 150°for between 10 minutes and 4 hours.
 22. A food composition made by themethod of claim 1, wherein the food composition is selected from thegroup consisting of reaction flavors, dairy alternative products, readyto mix beverages and beverage bases; extruded and extruded/puffedproducts; sheeted baked goods; texturized plant-based protein products;baked goods and baking mixes; granola; and soups/soup bases.
 23. A foodcomposition for human consumption, comprising a mixture of (a) amyceliated high-protein food product, wherein the myceliatedhigh-protein food product is at least 50% (w/w) protein on a dry weightbasis, wherein the myceliated high protein food product is derived froma plant source, wherein the myceliated high protein product ismyceliated by a fungal culture comprising Lentinula edodes, Agaricusblazeii, Pleurotus spp., Boletus spp., or Laetiporus spp. in a mediacomprising at least 50 g/L protein, and wherein the myceliated highprotein food product has reduced undesirable flavor and reducedundesirable aroma compared with a non-myceliated food product; and (b)an edible material.
 24. The food composition of claim 23, wherein thefood composition is selected from the group consisting of reactionflavors, dairy alternative products, ready to mix beverages and beveragebases; extruded and extruded/puffed products; sheeted baked goods;texturized plant-based protein products; baked goods and baking mixes;granola; and soups/soup bases.
 25. The food composition of claim 23,wherein the edible material is a starch, a flour, a grain, a lipid, acolorant, a flavorant, an emulsifier, a sweetener, a vitamin, a mineral,a spice, a fiber, a protein, nutraceuticals, sterols, isoflavones,lignans, glucosamine, an herbal extract, xanthan, a gum, a hydrocolloid,a starch, a preservative, a legume product, a food particulate, orcombinations thereof.
 26. The food composition of claim 23, wherein thefood composition is an extruded or an extruded/puffed materialcomprising pasta noodles, crisps, scoops, or a breakfast cereal, or thefood composition is a sheeted baked good such as tortillas, crackers, orpizza crust.
 27. The food composition of claim 23, wherein the foodcomposition is a texturized plant-based protein material.
 28. The foodcomposition of claim 27, wherein the mixture further comprises a starch,a flour, a grain, a lipid, a colorant, a flavorant, an emulsifier, asweetener, a vitamin, a mineral, a spice, a fiber, an enzyme, a proteinpowder, nutraceuticals, sterols, isoflavones, lignans, glucosamine, anherbal extract, xanthan, a gum, a hydrocolloid, a preservative, a legumeproduct, a food particulate, or combinations thereof.
 29. The foodcomposition of claim 27, wherein the mixture comprises about 45% peaprotein, 5% pea fiber, and about 50% myceliated high-protein material,w/w; or wherein the mixture comprises about 70% myceliated high-proteinmaterial, about 25% pea protein, about 5% pea fiber, w/w.
 30. The foodcomposition of claim 23, wherein the food composition is a bakedgood/baking mix comprising breads, cookies, muffins, pancakes, waffles,donuts, or brownies; or the food composition is a bar comprising abreakfast bar, nutritional bar or protein bar; or the food compositionis a granola product.
 31. The composition of claim 23 wherein the plantsource comprises pea, rice, or combinations thereof, and wherein thereduced undesirable flavor is a pea flavor or a bitterness flavor andthe reduced undesirable aroma is a beany aroma or a rice aroma.
 32. Amethod to prepare a textured plant-based meat analog or meat extender,comprising the steps of: (a) providing a myceliated high-protein foodproduct and at least one additional high-protein material, wherein themyceliated high-protein food product is at least 50% (w/w) protein on adry weight basis, wherein the myceliated high-protein food product isderived from pea and/or rice, wherein the myceliated high-proteinproduct is myceliated by an aqueous fungal culture, in a mediacomprising at least 50 g/L protein in liquid culture, and wherein themyceliated high-protein food product has reduced undesirable flavor andreduced undesirable aroma compared with a non-myceliated food product;and wherein the least one additional high-protein material comprises atleast 50% protein on a dry weight basis, (b) mixing the myceliatedhigh-protein food product and additional high-protein material, whereinthe myceliated high-protein food product is present at between about 5%and 90% on a dry weight basis compared with the additional high-proteinmaterial; (c) adding steam and/or water to the mixture; (d) extrudingthe mixture under heat and pressure to form the textured plant-basedmeat analog or meat extender.